ABSTRACT Burn patients who consumed alcohol before injury have worse clinical outcomes, including longer hospital stays, increased ventilator days, and more respiratory infections. Most alcohol consumers are binge drinkers and not chronic alcoholics, and binge drinking patterns fluctuate over the week, with consecutive days of drinking over the weekend followed by relative abstinence during the week. We used a murine model simulating this drinking pattern in the context of burn injury. Mice were given ethanol for 3 days, rested for 4 days, given ethanol for 3 more days, followed by a sham or 15% total body surface area full-thickness burn. We previously demonstrated that mice exposed to the combined insult exhibited respiratory dysfunction and 50% mortality, with those that succumbed to injury dying between 24 and 72 h, thus identifying a therapeutic intervention window. Our goal herein is to characterize inflammatory and respiratory parameters during this critical time frame. We saw that mice exposed to the combined insult had the highest circulating and pulmonary cytokine levels at 24 h, which were normalized by 72 h in survivors. Alveolar macrophage activation was observed at 24 h in burned mice, regardless of intoxication (P < 0.05). However, at 72 h, alveolar macrophages from intoxicated burned mice had elevated CD206, relative to controls (P < 0.05), indicative of an anti-inflammatory phenotype. Taken together, these findings suggest that although lung function and inflammation are normalized by 72 h, the alterations in alveolar macrophage phenotype shed light on a potential mechanism underlying increased infection susceptibility in intoxicated burn patients.

OBJECTIVE:To derive and validate a prediction model for the development of ARDS in burn-injured patients.SUMMARY BACKGROUND DATA:Burn injury carries the highest incidence of acute respiratory distress syndrome (ARDS) among all predisposing conditions, but few studies exist on risk factors in these patients. Studies employing biomarkers and clinical risk factors for predicting ARDS mortality have recently been examined but none exist for onset of ARDS nor in patients with burn injury.METHODS:This was a prospective multicenter study of 113 patients with isolated burn injury or inhalation injury. Clinical variables and plasma biomarkers representative of endothelial injury, epithelial injury, or inflammation were collected within 24 hours of admission. The most parsimonious model was chosen by considering discrimination, calibration, and model fit.RESULTS:Among the biomarkers measured in patients with burn injuries, a one-standard deviation increase in log-transformed levels of the A2 domain of von Willebrand factor in the first 24 hours was most strongly associated with the development of ARDS (OR 7.72; 95% CI: 1.64-36.28, P = 0.03). Of candidate models, a 3-variable model with %TBSA, inhalation injury, and von Willebrand factor-A2 had comparable discrimination to more complex models (area under the curve: 0.90; 95% CI 0.85-0.96). The 3-variable model had good model fit by Hosmer-Lemeshow test (P = 0.74) and maintained similar discrimination after accounting for performance optimism (Bootstrapped area under the curve: 0.90; 95% CI: 0.84-0.95).CONCLUSIONS:The 3-variable model with %TBSA, inhalation injury, and von Willebrand factor could be used to better identify at-risk patients for both the study and prevention of ARDS in patients with burn injury.

Cutaneous burn injury is one of the most devastating injuries one can obtain, with tissue damage extending beyond the skin wound to distal organs, including the gastrointestinal tract, liver, and lungs. Multiple organ failure is a leading cause of death after burn injury, resulting in excessive systemic and localized inflammation directly contributing to end organ damage. We postulated that the gut-liver-lung inflammatory axis underscores multiple organ failure in the context of burn injury and is hyper-activated when ethanol intoxication precedes burn. Mesenchymal stem cells (MSCs) are regenerative and anti-inflammatory, and MSC treatment has been shown to be beneficial in several immune disorders and injury models. Our objective was to determine whether intravenous infusion of exogenous bone marrow-derived MSCs could reduce post-burn and intoxication pulmonary, hepatic, and systemic inflammation. Vehicle- or ethanol- (1.6 g/kg) treated mice were subjected to sham or 15% total body surface area scald burn. One hour post-injury, mice were given 5 × 105 CFSE-labeled MSCs or phosphate-buffered saline intravenously (i.v.) and were euthanized 24 h later. We assessed circulating biomarkers of inflammation and liver damage, measured cytokine and chemokine production, and quantified apoptosis in lung and liver tissue. Compared to intoxicated and burned mice, those treated with MSCs had less cellularity, limited apoptosis, and a slight reduction in the pro-inflammatory cytokine interleukin-6 (IL-6) and the neutrophil chemokine, KC (CXCL1) in lung tissue. Mice with MSCs treatment had more dramatic anti-inflammatory effects on systemic and hepatic inflammation, as serum IL-6 levels were diminished by 43%, and il6 and kc expression in liver tissue were markedly reduced, as were biomarkers of liver damage, aspartate transaminase (AST) and alanine transaminase (AST), compared with intoxicated and burned mice. Taken together, our results suggest intravenous MSCs treatment can diminish systemic inflammation, lessen hepatic damage, and decrease liver and lung apoptosis and inflammation, indicating MSCs as a novel therapy for restoring homeostasis of multiple organ systems in intoxicated burn patients.

This prospective study aimed to address changes in inflammatory response between different aged populations of patients who sustained burn and inhalation injury. Plasma and bronchoalveolar lavage (BAL) samples were collected from 104 patients within 15h of their estimated time of burn injury. Clinical variables, laboratory parameters, and immune mediator profiles were examined in association with clinical outcomes. Older patients were at higher odds for death after burn injury (odds ratio (OR)=7.37 per 10years, p=0.004). In plasma collected within 15h after burn injury, significant increases in the concentrations of interleukin 1 receptor antagonist (IL-1RA), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), granulocyte colony-stimulating factor (G-CSF), interferon-gamma-induced protein 10 (IP-10) and monocyte chemoattractant protein 1 (MCP-1) (p<0.05 for all) were observed in the ≥65 group. In the BAL fluid, MCP-1 was increased three-fold in the ≥65 group. This study suggests that changes in certain immune mediators were present in the older cohort, in association with in-hospital mortality.

In this paper, we develop a new technique to study the optimal convergence orders of collocation methods for Volterra functional integral equations with vanishing delays on quasi-geometric meshes. Basing on a perturbation analysis, we show that for m collocation points, the global convergence order of the collocation solution is only m. However, the collocation solution may exhibit superconvergence with order m+1 at the collocation points. In particular, the local convergence order may attain 2m−1 at the nodes, provided that the collocation is based on the m Radau II points. Finally, some numerical examples are performed to verify our theoretical results.

OBJECTIVE:To evaluate disease activity statuses' (DAS') impact on systemic lupus erythematosus (SLE) outcomes. MATERIALS AND METHODS:Four DAS were defined: remission off-therapy: SLE Disease Activity Index (SLEDAI)=0, no prednisone or immunosuppressive drugs (IS); remission on-therapy: SLEDAI=0, prednisone ≤5 mg/day and/or IS (maintenance); low (L) DAS: SLEDAI ≤4, prednisone ≤7.5 mg/day and/or IS (maintenance); non-optimally controlled: SLEDAI >4 and/or prednisone >7.5 mg/day and/or IS (induction). Antimalarials were allowed in all. Predefined outcomes were mortality, new damage (increase of at least one Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) damage index (SDI) point) and severe new damage (increase of at least 3 SDI points). Univariable and multivariable Cox regression models were performed to define the impact of DAS, as time-dependent variable, on these outcomes. RESULTS:1350 patients were included, 79 died during follow-up, 606 presented new and 177 severe new damage. In multivariable analyses, remission (on/off-therapy) was associated with a lower risk of new (HR 0.60; 95% CI 0.43 to 0.85), and of severe new damage (HR 0.32; 95% CI 0.15 to 0.68); low disease activity status (LDAS) was associated with a lower risk of new damage (HR 0.66; 95% CI 0.48 to 0.93) compared with non-optimally controlled. No significant effect on mortality was observed. CONCLUSIONS:Remission was associated with a lower risk of new and severe new damage; LDAS with a lower risk of new damage after adjusting for other damage confounders.

Dermal burn injury causes profound physiological derangements. Respiratory failure is a primary cause of morbidity and mortality after burn injury, in part, because of excessive and prolonged release of local and systemic proinflammatory mediators. Clinical and preclinical evidence suggests histone deacetylases (HDACs) are key mediators of inflammatory responses. The study objective was to explore the effects of dermal burn injury on pulmonary HDAC activity, identify specific lung HDAC(s) altered by burn, and characterize histone lysine acetylation status. Mice were subjected to a 15% total body surface area scald burn or a sham injury and euthanized 24 hours later. Whole lungs were harvested, or alveolar macrophages were isolated from bronchoalveolar lavage fluid. HDAC specific activity assays were performed, Western blots were run to analyze HDACs1, 2, 3, 4, and 10 or histone lysine acetylation levels, and HDAC1 and phosphorylated-HDAC1 levels and localization were examined by immunofluorescence. Burned mice had higher HDAC specific activity and increased HDAC1 levels compared with controls, but levels of other HDACs were comparable between groups. Burn injury increased levels of HDAC1 and phosphorylated-HDAC1 in bronchioles and alveolar sacs and was associated with global and specific diminished levels of histone H3 and histone H4 lysine acetylation. Our analyses reveal that pulmonary inflammation after burn injury may be modulated by epigenetic mechanisms involving HDACs because HDAC activity, HDAC1 expression and activity, and downstream histone acetylation were all altered after burn. Future studies will explore the role of HDAC inhibitors in reversing inflammatory defects and may ultimately lead to new treatment interventions for burn patients.

Shortly after brain death, there is a catecholamine storm with up-regulation of pro-inflammatory cytokines. The impact of this pro-inflammatory environment on transplant outcomes has not been well-described. Donor factors are known to potentially contribute to the development of primary graft dysfunction (PGD). Our hypothesis is that organs from a donor with an increased pro-inflammatory state will result in an increased rate of primary graft dysfunction in the recipient following lung transplant. Serum and bronchoalveolar lavage (BAL) fluid were collected from 41 consecutive lung allograft donors. Multiplex bead array analysis was performed on serum and BAL supernatant. Clinical data was collected on the donors. Recipient PGD scores were obtained according to ISHLT guidelines with severe PGD designated as PGD grade 3 at 48 or 72 hours. 27 different cytokine concentrations were measured in the serum and BAL. There was no association between serum systemic inflammation in the donor and subsequent development of PGD. Case-matched controls were assessed with a Wilcoxon signed rank test comparing those with severe PGD (N=5) to those with no PGD (N=5). There remained no difference in donor serum cytokine concentrations in those without PGD and those who developed severe PGD. A correlation analysis looking at the relationship between serum cytokines and BAL cytokines found that IL-4 had a negative correlation (-0.52, p=0.0005). IL-6 and G-CSF trended to a positive correlation (0.3, p=0.06 and 0.25, p=0.12, respectively). There was no association between circulating biomarkers of systemic inflammation in donors prior to explantation of the lungs and subsequent development of PGD in the lung transplant recipients. Serum cytokines did not seem to strongly correlate with measured BAL cytokines, suggesting that the measured systemic inflammation in lung donors may be due to other non-pulmonary causes.

Dendritic cells (DCs) are a critical component of anti-tumor immunity due to their ability to induce a robust immune response to antigen (Ag). Alcohol was previously shown to reduce DC ability to present foreign Ag and promote pro-inflammatory responses in situations of infection and trauma. However the impact of alcohol exposure on generation of an anti-tumor response, especially in the context of generation of an immune vaccine has not been examined. In the clinic, DC vaccines are typically generated from autologous blood, therefore prior exposure to substances such as alcohol may be a critical factor to consider regarding the effectiveness in generating an immune response. In this study, we demonstrate for the first time that ethanol differentially affects DC and tumor Ag-specific T cell responses depending on sex. Signaling pathways were found to be differentially regulated in DC in females compared to males and these differences were exacerbated by ethanol treatment. DC from female mice treated with ethanol were unable to activate Ag-specific cytotoxic T cells (CTL) as shown by reduced expression of CD44, CD69, and decreased production of granzyme B and IFNγ. Furthermore, although FOXO3, an immune suppressive mediator of DC function, was found to be upregulated in DC from female mice, ethanol related suppression was independent of FOXO3. These findings demonstrate for the first time differential impacts of alcohol on the immune system of females compared to males and may be a critical consideration for determining the effectiveness of an immune based therapy for cancer in patients that consume alcohol.

The aim of this study was to assess the prevalence and associated features of cutaneous manifestations in patients with systemic lupus erythematosus (SLE) as well as to evaluate whether cutaneous manifestations are predictors of the occurrence of other clinical manifestations.

Severe primary graft dysfunction (PGD) is a serious cause of morbidity and mortality following lung transplantation. Donor factors can contribute to the risk of the recipient developing PGD. Brain death is known to cause an inflammatory state in the donor. Assessment of airway inflammation in the donor allograft and effect on development of PGD has been sparsely evaluated, but such information is important in the age of ex-vivo lung perfusion. Our hypothesis is that increased airway inflammation in the lung allograft prior to explant results in an increased risk of primary graft dysfunction in the recipient following lung transplant. Bronchoalveolar lavage (BAL) fluid was collected from 41 consecutive lung allograft donors just prior to cross-clamp and explant. Multiplex bead array analysis was performed on BAL supernatant. Clinical data was collected on donors and recipients including the development of PGD. PGD was defined according to the ISHLT guidelines, and severe PGD was designated if grade 3 was present at 48 or 72 hours post-lung transplant. Differences in cytokine concentrations were analyzed with a Wilcoxon rank sum test. Concentrations of several cytokines were elevated in the lung allograft prior to explant from the donor, and whose subsequent recipients went on to developed PGD. Relevant cytokines are shown in Figure 1 below, including IL-6, IL-8, IL-12, and VEGF. MCP-1 was also elevated [1455 (IQR 1536-4157) in those with PGD 0-2 versus 4167 (IQR 713.6-10,910) in those with PGD 3], p=0.02. Additional cytokines analyzed but did not differ included TNF-α, IP-10, IFN-γ, G-CSF, IL-17, IL-10, and IL-1β. Airway concentrations of IL-6, IL-8, IL-12, MCP-1, and VEGF were significantly elevated in allografts of donors whose recipients went on to develop PGD following lung transplant. Evaluation of these airway biomarkers might yield diagnostic testing to distinguish lungs which may benefit from ex-vivo perfusion.

In this study, the role and fate of AMs were examined in pulmonary inflammation after intoxication and injury. Clinical evidence has revealed that half of all burn patients brought to the emergency department are intoxicated at the time of injury. This combined insult results in amplified neutrophil accumulation and pulmonary edema, with an increased risk of lung failure and mortality, relative to either insult alone. We believe that this excessive pulmonary inflammation, which also parallels decreased lung function, is mediated in part by AMs. Restoration of lung tissue homeostasis is dependent on the eradication of neutrophils and removal of apoptotic cells, both major functions of AMs. Thirty minutes after binge ethanol intoxication, mice were anesthetized and given a 15% total body surface area dorsal scald injury. At 24 h, we found a 50% decrease in the total number of AMs (P < 0.05) and observed a proinflammatory phenotype on the remaining lung AMs. Loss of AMs paralleled a 6-fold increase in the number of TUNEL+ lung apoptotic cells (P < 0.05) and a 3.5-fold increase in the percentage of annexin V+ apoptotic cells in BAL (P < 0.05), after intoxication and injury, relative to controls. In contrast to the reduction in the number of cells, AMs from intoxicated and injured mice had a 4-fold increase in efferocytosis (P < 0.05). In summary, these data suggest that loss of AMs may delay resolution of inflammation, resulting in the pulmonary complications and elevated mortality rates observed in intoxicated and burn-injured patients.

Alcohol intoxication is involved in 50% of all burn injuries and results in an increased risk of lung infection and mortality, relative to non-drinking patients. Experimental models of intoxication, burn and Pseudomonas aeruginosa (PA) intratracheal (i.t.) infection have revealed amplified lung inflammation, relative to controls. Since lung inflammation is mediated in part by alveolar macrophages (AMs), we chose to examine AMs in this combined injury model. Mice were subjected to ethanol 30 min prior to a 15% total body surface area scald injury, followed by an i.t. PA infection (3,000 CFUs). At 24 h, bronchoalveolar lavage cells were analyzed by flow cytometry for absolute numbers of AMs. Relative to shams, burn and infected mice had 60% less AMs (p<0.05), while combined injured, intoxicated, burn and infected mice had 80% less AMs (p<0.05). Between burn groups, combined injury had 60% less AMs, relative to burn and infection alone. Lungs from shams revealed 3 apoptotic cells per low powered field. This was increased 2-fold after burn and infection and by 8-fold with combined injury (p<0.05). Combined injured mice also had 5-fold more apoptotic cells than burn and infection alone (p<0.05). These data suggest a decrease in AM numbers may be due to an increase in AM apoptosis. Overall, the loss of AMs may contribute to the exacerbated lung inflammation observed with combined injury. [R01AA012034 (EJK), T32 AA013527 (EJK), F31 AA022566 (JAI), and the Falk Foundation (EJK)].

Clinical data indicate that cutaneous burn injuries covering greater than 10% of the total body surface area are associated with significant morbidity and mortality, in which pulmonary complications, including acute respiratory distress syndrome (ARDS), contribute to nearly half of all patient deaths. Approximately 50% of burn patients are intoxicated at the time of hospital admission, which increases days on ventilators by 3-fold, and doubles the length of hospitalization, compared to non-intoxicated burn patients. The most common drinking pattern in the United States is binge drinking, where an individual rapidly consumes alcoholic beverages (4 for women, 5 for men) in 2 h. An estimated 38 million Americans binge drink, often several times per month. Experimental data demonstrate that a single binge-ethanol exposure, prior to scald injury, impairs innate and adaptive immune responses, thereby enhancing infection susceptibility and amplifying pulmonary inflammation, neutrophil infiltration, and edema, and is associated with increased mortality. Since these characteristics are similar to those observed in ARDS burn patients, our study objective was to determine whether ethanol intoxication and burn injury and the subsequent pulmonary congestion affect physiological parameters of lung function, using non-invasive and unrestrained plethysmography in a murine model system. Furthermore, to mirror young adult binge-drinking patterns, and to determine the effect of multiple ethanol exposures on pulmonary inflammation, we utilized an episodic binge-ethanol exposure regimen, where mice were exposed to ethanol for a total of 6 days (3 days ethanol, 4 days rest, 3 days ethanol) prior to burn injury. Our analyses demonstrate mice exposed to episodic binge ethanol and burn injury have higher mortality, increased pulmonary congestion and neutrophil infiltration, elevated neutrophil chemoattractants, and respiratory dysfunction, compared to burn or ethanol intoxication alone. Overall, our study identifies plethysmography as a useful tool for characterizing respiratory function in a murine burn model and for future identification of therapeutic compounds capable of restoring pulmonary functionality.