1. Urinary biomarkers and cytokine response in recent drinkers compared to non-recent drinkers after binge ethanol dosing and phytohemagglutinin-M (PHA) stimulation M. Afshar, J. Hasday, G. Netzer, G.S. Smith, Pulmonary and Critical Care Medicine, Shock Trauma and Anesthesiology Research (STAR) – Organized Research Center, University of Maryland Medical Center, Baltimore, MD, USA Ethanol intoxication is present in up to 50% of trauma patients. Severe injury may activate the systemic inflammatory response syndrome (SIRS). How binge ethanol affects patients with SIRS is unclear. We performed a pilot study evaluating binge ethanol consumption prior to ex-vivo endotoxin challenge and its effects on immune responsiveness. Urinary EtG (Ethyl Glucuronide) and EtS (Ethyl Sulfate) were used to compare subjects consuming ethanol 24–48 h prior to ethanol dosing (recent drinkers) to those who did not (non-recent drinkers). Ten healthy human volunteers characterized as low to moderate drinkers provided urine samples prior to and 24 h after ethanol dosing (vodka drink over 20 min: males 0.89 g/kg, females 0.81 g/kg). Blood alcohol content (BAC) and heparinized blood was collected prior to dosing, 20 min, 2 h, and 5 h after dosing. Aliquots of blood were diluted 1:1 RPMI 1640 medium and incu- bated for 24 h with 0.6–6% PHA. Culture supernatants were collected by centrifugation and cytokine levels were measured by ELISA. Four subjects were recent drinkers and had elevated pre-dosing EtG and EtS levels (23,921 Æ 35,178 ng/ml and 4418 Æ 6262 ng/ml), and the 6 non-recent drinkers had no measurable levels. The mean peak BAC was 142 Æ 36 mg/ dl. No difference in BAC between recent and non-recent drinkers was found at the three sampling points. No differences in cytokine expressions by drinking type were found in PHA-stimulated blood prior to binge ethanol dosing. Similar elevations of PHA-stimulated IFN-g, and IL-8 levels occurred after binge ethanol dosing. IL-2 expression was greater in recent drinkers after binge (p ¼ 0.03) compared to non-recent drinkers. This suggests that recent drinkers of ethanol may have a modulated immune response after binging compared to non-recent drinkers. EtG and EtS are useful to identify recent drinkers who have no detectable BAC. Studies investigating the immunomodulatory effects of ethanol exposure after injury or SIRS should further delineate exposure to include recent drinkers utilizing EtG and EtS. 2. Role of microRNA-155 in alcoholic liver disease S. Bala, T. Csak, J. Zatsiorsky, D. Catalano, K. Kodys, G. Szabo, Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA MicroRNAs (miRNAs) are small non-coding RNAs that emerged as new crucial regulatory players of all cellular processes. miRNA-155 is a multi- functional miRNA that regulates various immune and non-immune bio- logical processes. Previously, we showed the induction of miR-155 in alcoholic liver disease (ALD) where it contributes to inflammation via regulating TNF alpha. To further elucidate its biological role in ALD, we employed miR-155 deficient (KO) mice. Female wild type (C57/BL6J) or miR-155 KO or TLR4 KO mice (n ¼ 8–10) were fed the Lieber-DeCarli diet containing either ethanol or control diet for 5 weeks. Our results indicate that miR-155 KO mice are protective from alcohol-induced liver inflam- mation and gut permeability. A significant attenuation in liver TNF alpha, IL-1beta and MCP1 (mRNA and protein) was found in alcohol-fed miR-155 KO mice compared to wild type mice treated with or without LPS (0.5 mg/ kg for 3 h prior to conclusion of the experiment). Surprisingly, there was no increase in plasma endotoxin levels in miR-155 deficient mice after chronic alcohol feeding, indicative of intact gut epithelium. Further, a partial, but significant decrease in fat accumulation was observed in alcohol-fed miR-155 KO mice compared to wild type mice. Alcohol feeding resulted in a significant induction of oxidative stress (measured by TBAR assay) in wild type mice and this increase was prevented in miR-155 KO mice. Interestingly, alcohol-induced decrease in hepatic miR-122 levels observed in wild type mice was prevented in miR-155 KO mice, suggesting miR-155 plays a causal role in alcoholic liver injury. Our results also indicate that alcohol induces miR-155 via TLR4 pathway as TLR4 KO mice in contrast to wild type mice showed no increase in miR-155 after alcohol feeding. In summary, our results suggest that miR-155 plays an essential role in the pathogenesis of ALD and therapeutic inhibition of this miRNA might be an attractive strategy to ameliorate ALD (Supported by NIH AA020744 [GS]). 3. Chronic plus binge ethanol feeding synergistically induces neutrophil infiltration and liver injury: A critical role for E-selectin A. Bertola, O. Park, B. Gao, Laboratory of Liver Diseases, NIAAA, NIH, Bethesda, MD, USA Our laboratory has recently developed an improved mouse model of alcoholic liver disease (the NIAAA model). This new model incorporates both chronic and binge ethanol feeding (10 days ad libitum feeding with ethanol containing liquid diet plus a single binge of ethanol (5 g/kg) by oral gavage), which closely resembles the drinking pattern and acute-on-chronic liver injury of many alcoholic hepatitis patients. In the present study, we investigated the mechanisms underlying the synergistic effect of chronic plus binge ethanol feeding on liver injury. We found that chronic plus binge ethanol feeding synergistically upregulated the hepatic expression of proinflammatory cytokines (IL-1b, TNF-a) and chemokines (MCP-1, MIP-2), and induced significant neutrophil accumulation in the liver compared to chronic or binge feeding alone. In vivo depletion of neutrophils through administration of the anti-Ly6G antibody markedly reduced chronic-binge ethanol feeding-induced liver injury. Real-time PCR analyses revealed that the hepatic expression of E-selectin was 10-fold upregulated, whereas the expression of other neutrophil infiltration-related adhesion molecules (e.g., P-selectin, ICAM-1, and VCAM-1) was slightly up- or down-regulated in this chronic-binge ethanol feeding model. The genetic deletion of E-selectin prevented chronic-binge ethanol feeding-induced hepatic neutrophil infil- tration and elevation of serum transaminases without affecting ethanol- induced liver steatosis. In addition, E-selectin-deficient mice showed reduced hepatic expression of several proinflammatory cytokines, chemo- kines, and adhesion molecules compared to wild-type mice after chronic- binge ethanol feeding. Finally, the expression of E-selectin was highly upregulated in human alcoholic fatty livers but not in alcoholic cirrhotic livers. Conclusions: Chronic plus binge ethanol feeding upregulates the hepatic expression of proinflammatory cytokines, followed by the induction of E-selectin. Elevated E-selectin plays an important role in neutrophil recruitment, inflammation, and injury in the alcoholic liver injury induced by chronic plus binge ethanol feeding in mice and may also contribute to the pathogenesis of early stages of human alcoholic liver disease. 4. Alcohol consumption amplifies immune suppression induced by ultraviolet radiation R.M. Brand, J.M. Stottlemyer, M.C. Paglia, L.D. Falo, Jr., Departments of Dermatology and Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA The growing health care burden from increasing rates of nonmelanoma skin cancer (NMSC), the most common malignancy in the United States, highlights the need for better implementation and/or development of more effective preventive and mitigating strategies. Results from epidemiological studies have shown that individuals who regularly drink alcohol have greater rates of NMSC. Since both alcohol consumption and exposure to UV radiation independently cause immune suppression, we hypothesize that these factors will have a synergistic effect on immune suppression. Female C57/BL6 mice were either given alcohol chronically or acutely prior to UVR exposure using Westinghouse FS72T12/UVB lamps at a dose of 100 mJ/cm 2 daily for 4 days to test for alcohol-UV interactions. Ears were covered with black electrical tape prior to UV exposure to ensure systemic immune suppression was measured. On day 5, 1% of the contact allergen oxazolone was applied to the belly. On day 10, 1% oxazolone was applied to the ears and swelling was measured 24 h later. UV, chronic alcohol or acute alcohol exposures each inhibited immune responses. Mice given both ETOH and UV demonstrate greater immune suppression than those exposed to either alone. This synergism occurs when ETOH was ingested chronically with the Lieber DeCarli Diet, by gavage at 5 g/kg given 30 min prior to each of the four UV doses or only once, prior to the first UV dose. To further examine the interactions between alcohol consumption and UV radiation in the induction of NMSC, we measured early markers of UV damage in the skin, including melanin synthesis, DNA damage, apoptosis and antioxidant depletion. UV-induced melanin is inhibited by acute alcohol consumption prior to UV exposure and overall skin damage is more extensive after both alcohol and UV than either alone. These results demonstrate that alcohol consumption and UV exposure act synergistically to increase severity of systemic immune suppression and local skin damage. These results provide potential mechanisms to explain the observation that alcohol consumers have a higher incidence of NMSC and warrants further investigation (Sup- ported by NIH K01 AA017907). Abstracts / Alcohol 47 (2013) 567–576 568