Urinary biomarkers and cytokine response in recent drinkers compared to non-recent drinkers after binge ethanol dosing and phytohemagglutinin-M (PHA) stimulation

s / Alcohol 47 (2013) 567–576 568 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 incubated 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 35,178 ng/ml and 4418 6262 ng/ml), and the 6 non-recent drinkers had no measurable levels. The mean peak BAC was 142 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 1⁄4 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 multifunctional miRNA that regulates various immune and non-immune biological 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 1⁄4 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 inflammation 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,