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Telomere shortening is usually considered a biomarker of ageing. Harmful alcohol use promotes accelerated biological ageing and alcohol use disorders (AUDs) are associated with short telomere length (TL). This study was conducted to examine the relationship of TL to AUD and determine whether single nucleotide polymorphisms (SNPs) in TERC and TERT modulate this association. For this purpose, we genotyped TERC SNPs rs2293607, rs12696304, and rs16847897 and TERT SNPs rs2735940, rs2736100, and rs2736098 in 308 male patients with AUD and 255 sex-matched healthy controls and measured TL in a subset of 99 patients and 99 controls paired by age and smoking status. Our results showed that the mean TL was shorter in patients with AUD than in controls. The area under the ROC curve was 0.70 (P < 0.001). The GG genotype of TERC rs2293607 was more common among patients with AUD than among controls (9.8% vs. 5.1%; P = 0.038). No difference was found for the other SNPs. Carriers of the GG genotype of rs2293607 had shorter telomeres than did allele A carriers. In conclusion, patients with AUD had shorter telomeres. Genetic susceptibility to telomere shortening through the rs2293607 SNP is associated with a greater risk of AUD.

Resumen Introduccion El consumo de alcohol induce una respuesta inflamatoria mediada por los receptores de tipo Toll 4 (TLR4) y el factor nuclear (NF)-?B, originando dano organico. Algunos micro-ARN (miARN) modulan la respuesta inflamatoria mediante retroalimentacion negativa de mediadores como las interleucinas (IL). Asi pues, polimorfismos en los genes de algunas IL localizados cerca de las dianas de los miARN podrian modificar el riesgo de dano organico inducido por el alcohol. Este estudio analizo la posible relacion entre el alcoholismo o la enfermedad hepatica alcoholica (EHA) y los polimorfismos IL12B 2124 G>T (rs1368439), IL16 5000 C >T (rs1131445), IL1R1 3114 C>T (rs3917328) y NFKB1 3400 A>G (rs4648143). Pacientes y metodos Se incluyeron 301 pacientes alcoholicos varones y 156 voluntarios sanos varones. Los polimorfismos fueron genotipados mediante discriminacion alelica utilizando el sistema de PCR TaqMan®. Se compararon las frecuencias alelicas y genotipicas entre grupos y se realizo un analisis de regresion logistica para dilucidar el modelo de herencia. Resultados El analisis del polimorfismo de IL1R1 (rs3917328) mostro que la proporcion de portadores del alelo T (genotipos CT y TT) era mayor en los controles sanos (9,7%) que en pacientes alcoholicos (6,5%, p = 0,042). Sin embargo el analisis de regresion logistica no mostro resultados significativos. No se encontraron diferencias significativas entre grupos con respecto al resto de polimorfismos estudiados. Conclusiones Nuestro estudio describe, por primera vez, las frecuencias esperadas de polimorfismos en regiones diana de miARN en pacientes alcoholicos con y sin EHA. Seran necesarios nuevos estudios para aclarar la relevancia de estos polimorfismos en el desarrollo de alcoholismo o EHA.

Heavy binge drinking in adolescence can cause long-term cognitive and behavioral dysfunctions. Recent experimental evidence indicates the participation of immune system activation in the effects of ethanol in the adolescent brain and suggests gender differences. The present study aims to assess plasma cytokine and chemokine levels in male and female adolescents and young adults during acute alcohol intoxication and to correlate these results with the toll-like receptor 4 (TLR4) response. The potential role of the TLR4 signaling response was also assessed in plasma and prefrontal cortex (PFC) of adolescent wild-type and TLR4-knockout male and female mice with binge ethanol treatment. The results showed that alcohol intoxication increased the plasma levels of several cytokine and chemokine [interferon-γ, interleukin (IL)-10, IL-17A, IL-1β, IL-2, IL-4, IL-6, IL-8, fractalkine, monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1α (MIP-1α)] and the upregulation of TLR4 mRNA levels occurred in intoxicated females, while elevation of colony-stimulating factor was only observed in the plasma of males. In wild-type female adolescent mice, intermittent ethanol treatment increased the levels of several cytokines (IL-17A and IL-1β) and chemokines (MCP-1, MIP-1α and fractalkine) in PFC and in serum (IL-17A, MCP-1 and MIP-1α), but significant differences in the fractalkine levels in PFC were observed only in male mice. No changes in serum or prefrontal cortex cytokine and chemokine levels were noted in ethanol-treated male or female TLR4-knockout mice. Our findings revealed that females are more vulnerable than males to inflammatory effects of binge ethanol drinking and suggested that TLR4 is an important target of ethanol-induced inflammation and neuroinflammation in adolescence.

Current studies evidence the role of miRNAs in extracellular vesicles (EVs) as key regulators of pathological processes, including neuroinflammation and neurodegeneration. As EVs can cross the blood&ndash, brain barrier, and EV miRNAs are very stable in peripheral circulation, we evaluated the potential gender differences in inflammatory-regulated miRNAs levels in human and murine plasma EVs derived from alcohol-intoxicated female and male adolescents, and whether these miRNAs could be used as biomarkers of neuroinflammation. We demonstrated that while alcohol intoxication lowers anti-inflammatory miRNA (mir-146a-5p, mir-21-5p, mir-182-5p) levels in plasma EVs from human and mice female adolescents, these EV miRNAs increased in males. In mice brain cortices, ethanol treatment lowers mir-146a-5p and mir-21-5p levels, while triggering a higher expression of inflammatory target genes (Traf6, Stat3, and Camk2a) in adolescent female mice. These results indicate, for the first time, that female and male adolescents differ as regards the ethanol effects associated with the inflammatory-related plasma miRNAs EVs profile, and suggest that female adolescents are more vulnerable than males to the inflammatory effects of binge alcohol drinking. These findings also support the view that circulating miRNAs in EVs could be useful biomarkers for screening ethanol-induced neuroinflammation and brain damage in adolescence.

[EN]MicroRNAs (miRNAs) are small non-coding RNAs that regulate multiple physiological and pathological functions through the modulation of gene expression at the post-transcriptional level. Accumulating evidence has established a role for miRNAs in the development and pathogenesis of liver disease. Specifically, a large number of studies have assessed the role of miRNAs in alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), two diseases that share common underlying mechanisms and pathological characteristics. The purpose of the current review is to summarize and update the body of literature investigating the role of miRNAs in liver disease. In addition, the potential use of miRNAs as biomarkers and/or therapeutic targets is discussed. Among all miRNAs analyzed, miR-34a, miR-122 and miR-155 are most involved in the pathogenesis of NAFLD. Of note, these three miRNAs have also been implicated in ALD, reinforcing a common disease mechanism between these two entities and the pleiotropic effects of specific miRNAs. Currently, no single miRNA or panel of miRNAs has been identified for the detection of, or staging of ALD or NAFLD. While promising results have been shown in murine models, no therapeutic based-miRNA agents have been developed for use in humans with liver disease.

BACKGROUND: Alcohol binge drinking is one of the most common patterns of excessive alcohol use and recent data would suggest that histone deacetylases (HDACs) gene expression profiling could be useful as a biomarker for psychiatric disorders. METHODS: This study aimed to characterize the gene expression patterns of Hdac 1-11 in samples of rat peripheral blood, liver, heart, prefrontal cortex, and amygdala following repeated binge alcohol consumption and to determine the parallelism of Hdac gene expression between rats and humans in peripheral blood. To accomplish this goal, we examined Hdac gene expression following 1, 4, or 8 alcohol binges (3 g/kg, orally) in the rat, in patients who were admitted to the hospital emergency department for acute alcohol intoxication, and in rats trained in daily operant alcohol self-administration. RESULTS: We primarily found that acute alcohol binging reduced gene expression (Hdac1-10) in the peripheral blood of alcohol-naïve rats and that this effect was attenuated following repeated alcohol binges. There was also a reduction of Hdac gene expression in the liver (Hdac2,4,5), whereas there was increased expression in the heart (Hdac1,7,8) and amygdala (Hdac1,2,5). Additionally, increased blood alcohol concentrations were measured in rat blood at 1 to 4 hours following repeated alcohol binging, and the only group that developed hepatic steotosis (fatty liver) were those animals exposed to 8 alcohol binge events. Finally, both binge consumption of alcohol in humans and daily operant alcohol self-administration in rats increased Hdac gene expression in peripheral blood. CONCLUSIONS: Our results suggest that increases in HDAC gene expression within the peripheral blood are associated with chronic alcohol consumption, whereas HDAC gene expression is reduced following initial exposure to alcohol. This work was supported by The European Foundation for Alcohol Research (to JAL-M, FRdF, RM, RN, and MPV), the Fondo de Investigación Sanitaria (Red de Trastornos Adictivos, FEDER, RD12/0028/0015 to JAL-M, RD12/0028/001 to FRdF, RD12/0028/023 to RM, RD12/0028/0014 to RN, RD12/0028/1021 to MPV, RD12/0028/0008 to F-JL; and PI10/01692 to MM), and Ministerio de Ciencia e Innovación (SAF2011-26818 to JAL-M)

[Background]: Although decreased counts of peripheral blood (PB) B cells—associated with an apparently contradictory polyclonal hypergammaglobulinemia—have been reported in chronic alcoholism, no information exists about the specific subsets of circulating B cells altered and their relationship with antibody production. Here, we analyzed for the first time the distribution of multiple maturation-associated subpopulations of PB B cells in alcoholism and its potential relationship with the onset of liver disease. [Methods]: PB samples from 35 male patients—20 had alcoholic hepatitis (AH) and 15 chronic alcoholism without liver disease (AWLD)—were studied, in parallel to 19 male healthy donors (controls). The distribution of PB B-cell subsets (immature/regulatory, naïve, CD27− and CD27+ memory B lymphocytes, and circulating plasmablasts of distinct immunoglobulin—Ig—isotypes) was analyzed by flow cytometry. [Results]: Patients with AH showed significantly decreased numbers of total PB B lymphocytes (vs. controls and AWLD), at the expense of immature, memory, and, to a lesser extent, also naïve B cells. AWLD showed reduced numbers of immature and naïve B cells (vs. controls), but higher PB counts of plasmablasts (vs. the other 2 groups). Although PB memory B cells were reduced among the patients, the percentage of surface (s)IgA+ cells (particularly CD27−/sIgA+ cells) was increased in AH, whereas both sIgG+ and sIgA+ memory B cells were significantly overrepresented in AWLD versus healthy donors. Regarding circulating plasmablasts, patients with AH only showed significantly reduced counts of sIgG+ cells versus controls. In contrast, the proportion of both sIgA+ and sIgG+ plasmablasts—from all plasmablasts—was reduced in AH and increased in AWLD (vs. the other 2 groups). [Conclusions]: AH and AWLD patients display a significantly reduced PB B-cell count, at the expense of decreased numbers of recently produced immature/regulatory B cells and naïve B cells, together with an increase in Ig-switched memory B lymphocytes and plasmablasts, particularly of IgA+ cells., This study was supported by the Plan Nacional sobre Drogas, grant number 2007/020 to F-J. L., Ministerio de Sanidad y Consumo, Madrid, Spain and the Red de Trastornos Adictivos of Instituto de Salud Carlos III—FONDOS FEDER with expedient numbers RD06/0001/0004 and RD12/0028/0008 to FJL.

Introduction Alcohol consumption promotes inflammation through the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB pathway, leading to organic damage. Some micro-RNA (miRNA) molecules modulate this inflammatory response by downregulating TLR4/NF-κB pathway mediators, like interleukins (ILs). Thus, polymorphisms within IL genes located near miRNA binding sites could modify the risk of ethanol-induced damage. The present study analyzed potential relationships between alcoholism or alcoholic liver disease (ALD) and IL12B 2124 G>T (rs1368439), IL16 5000 C>T (rs1131445), IL1R1 3114 C>T (rs3917328), and NFKB1 3400 A>G (rs4648143) polymorphisms. Patients and methods The study included 301 male alcoholic patients and 156 male healthy volunteers. Polymorphisms were genotyped using TaqMan ® PCR assays for allelic discrimination. Allele and genotype frequencies were compared between groups. Logistic regression analysis was performed to analyze the inheritance model. Results Analysis of the IL1R1 (rs3917328) polymorphism showed that the proportion of allele T carriers (CT and TT genotypes) was higher in healthy controls (9.7%) than in alcoholic patients (6.5%; p = .042). However, multivariable logistic regression analyses did not yield a significant result. No differences between groups were found for other analyzed polymorphisms. Conclusions Our study describes, for the first time, the expected frequencies of certain polymorphisms within miRNA-binding sites in alcoholic patients with and without ALD. Further studies should be developed to clarify the potential relevance of these polymorphisms in alcoholism and ALD development.

SummaryBackground Several studies have reported an association between alcoholic liver cirrhosis (ALC) or other forms of alcoholic liver disease (ALD) and the genetic variant rs738409 (C>G) in adiponutrin/patatin-like phospholipase domain-containing 3 gene (PNPLA3). Aim To evaluate the influence of this variant on ALC and other forms of ALD. Methods We performed a systematic review of previous studies on the relationship between rs738409 of PNPLA3 and ALD and meta-analysis was conducted in a random-effects model. Calculations of the odds ratios (ORs) and their confidence intervals (CIs), tests for heterogeneity and sensitivity analyses were performed. Results Database search identified 11 previous studies available for inclusion with a total of 3495 patients with ALD (2087 with ALC) and 5038 controls (4007 healthy subjects and 1031 alcoholics without ALD). Patients with ALC compared to controls had a significantly higher prevalence of the G allele when comparing GG vs. CC (OR 4.30, 95% CI 3.25–5.69; P

Aims: Prevalence of chronic hepatitis C virus (HCV) infection among alcoholics is thought to be higher than in the general population, although prevalence rates reported are quite variable. Our study is aimed to analyze HCV prevalence in a cohort of alcoholics and to perform a systematic review on this topic. Patients and methods: A total of 396 alcoholic patients consecutively referred to our Alcoholism Unit were included. HCV infection status and other clinical variables were recorded for each patient. Variables associated with HCV infection were analyzed by means of logistic regression. Additionally, we performed a systematic review focused on previous studies on this topic. Results: Among our alcoholic patients, 14 of them (3.53%) had chronic HCV infection. Variables independently associated with HCV infection were female gender, injection drug use (IDU) and the presence of alcoholic liver disease (ALD). Twenty-four studies analyzing HCV prevalence in alcoholic patients were included in our systematic review, showing prevalence rates of HCV infection ranging from 2.1 to 51% and an average weighted prevalence of 16.32%. Conclusion: In our series, the prevalence rate of chronic HCV infection among alcoholic patients is lower than previously reported, which is probably explained by the relatively low number of patients with ALD or IDU in our sample. Prevalence rates previously published are quite different and the presence of ALD and/or IDU can act as confounding factors for HCV prevalence among alcoholics.

Background\ud Genetic polymorphisms in the RAS gene family are associated with different diseases, which may include alcohol-related disorders. Previous studies showed an association of the allelic variant rs26907 in RASGRF2 gene with higher alcohol intake. Additionally, the rs61764370 polymorphism in the KRAS gene is located in a binding site for the let-7 micro-RNA family, which is potentially involved in alcohol-induced inflammation. Therefore, this study was designed to explore the association between these two polymorphisms and susceptibility to alcoholism or alcoholic liver disease (ALD).\ud \ud Methods\ud We enrolled 301 male alcoholic patients and 156 healthy male volunteers in this study. Polymorphisms were genotyped by using TaqMan® PCR assays for allelic discrimination. Allelic and genotypic frequencies were compared between the two groups. Logistic regression analysis was performed to analyze the inheritance model.\ud \ud Results\ud The A allele of the RASGRF2 polymorphism (rs26907) was significantly more prevalent among alcoholic patients with cirrhosis (23.2%) compared to alcoholic patients without ALD (14.2%). This difference remained significant in the group of patients with alcohol dependence (28.8% vs. 14.3%) but not in those with alcohol abuse (15.1% vs. 14.4%). Multivariable logistic regression analysis showed that the A allele of this polymorphism (AA or GA genotype) was associated with alcoholic cirrhosis both in the total group of alcoholics (odds ratio [OR]: 2.33, 95% confidence interval [CI]: 1.32–4.11; P = 0.002) and in the group of patients with alcohol dependence (OR: 3.1, 95% CI: 1.50–6.20; P = 0.001). Allelic distributions of the KRAS polymorphism (rs61764370) did not differ between the groups.\ud \ud Conclusions\ud To our knowledge, this genetic association study represents the first to show an association of the RASGRF2 G>A (rs26907) polymorphism with ALD in men, particularly in the subgroup of patients with AD. The findings suggest the potential relevance of the RAS gene family in alcoholism and ALD.

Heavy binge drinking in adolescence can cause long-term cognitive and behavioral dysfunctions. Recent experimental evidence indicates the participation of immune system activation in the effects of ethanol in the adolescent brain and suggests gender differences. The present study aims to assess plasma cytokine and chemokine levels in male and female adolescents and young adults during acute alcohol intoxication and to correlate these results with the toll-like receptor 4 (TLR4) response. The potential role of the TLR4 signaling response was also assessed in plasma and prefrontal cortex (PFC) of adolescent wild-type and TLR4-knockout male and female mice with binge ethanol treatment. The results showed that alcohol intoxication increased the plasma levels of several cytokine and chemokine [interferon-γ, interleukin (IL)-10, IL-17A, IL-1β, IL-2, IL-4, IL-6, IL-8, fractalkine, monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1α (MIP-1α)] and the upregulation of TLR4 mRNA levels occurred in intoxicated females, while elevation of colony-stimulating factor was only observed in the plasma of males. In wild-type female adolescent mice, intermittent ethanol treatment increased the levels of several cytokines (IL-17A and IL-1β) and chemokines (MCP-1, MIP-1α and fractalkine) in PFC and in serum (IL-17A, MCP-1 and MIP-1α), but significant differences in the fractalkine levels in PFC were observed only in male mice. No changes in serum or prefrontal cortex cytokine and chemokine levels were noted in ethanol-treated male or female TLR4-knockout mice. Our findings revealed that females are more vulnerable than males to inflammatory effects of binge ethanol drinking and suggested that TLR4 is an important target of ethanol-induced inflammation and neuroinflammation in adolescence.

Previous studies have suggested that the effect of naltrexone in patients with alcohol dependence may be moderated by genetic factors. In particular, the possession of the G allele of the A118G polymorphism of the µ-opioid receptor gene (OPRM1) has been associated with a better response to naltrexone, although controversial results have been reported. The aim of this paper is to combine previous findings by means of a systematic review and a meta-analysis. We retrieved studies on the relationship between A118G polymorphism in OPRM1 gene and response to treatment with naltrexone in patients with alcohol dependence by means of electronic database search. A meta-analysis was conducted using a random-effects model. Calculations of odds ratio (OR) and their confidence intervals (CI) and tests for heterogeneity of the results have been performed. Six previous studies have analyzed the role of A118G polymorphism in response to naltrexone for alcohol dependence. After meta-analysis, we found that naltrexone-treated patients carrying the G allele had lower relapse rates than those who were homozygous for the A allele (OR: 2.02, 95% CI 1.26-3.22; P = 0.003). There were no differences in abstinence rates. Our results support the fact that the G allele of A118G polymorphism of OPRM1 moderates the effect of naltrexone in patients with alcohol dependence. This genetic marker may therefore identify a subgroup of individuals more likely to respond to this treatment.

espanolINTRODUCCION: la presencia de anemia es un hallazgo frecuente entre los enfermos que ingresan en los servicios de Medicina Interna, aunque diferentes estudios han demostrado la influencia de la anemia sobre la calidad de vida, mortalidad y morbilidad en pacientes de edad avanzada, no se han realizado trabajos especificos en sujetos que cumplieran criterios estrictos de paciente pluripatologico (PPP). Nuestro objetivo fue analizar la prevalencia e importancia pronostica de la anemia en una cohorte de pacientes pluripatologicos. PACIENTES Y METODO: estudio prospectivo en el que se incluyeron una cohorte de pacientes pluripatologicos recogidos de forma consecutiva entre pacientes ingresados a lo largo de un ano; se estudio la presencia de anemia y se recogieron variables analiticas, sociodemograficas, clinicas y funcionales al ingreso y reevaluacion del estado vital y funcional al ano. Se analizaron las diferencias en la presencia o no de anemia y en la mortalidad al ano, incluyendo como factores de confusion variables sociodemograficas y de comorbilidad mediante analisis univariante y de regresion logistica multivariante. RESULTADOS: fueron incluidos 234 pacientes con edad media (desviacion estandar) de 80,3 (9,2) anos, 104 (44,5%) mujeres y 130 (55,5%) hombres, de los que presentaba anemia el 60,6%. Los tipos principales de anemia fueron anemia ferropenica (47,5% del total de anemias) y de procesos cronicos (29,4%). La mortalidad global fue del 56% en los pacientes con anemia y del 35,2% en los pacientes sin anemia (p = 0,003). Tras el analisis multivariante de regresion logistica, las variables asociadas de forma independiente y significativa con la mortalidad a un ano fueron la presencia de anemia, una puntuacion > 3 en el indice de Charlson y la insuficiencia respiratoria cronica. CONCLUSIONES: la importancia pronostica de la anemia en pacientes pluripatologicos, resultado no comunicado previamente, refuerza la importancia de una valoracion global y completa de estos pacientes. EnglishINTRODUCTION: The presence of anemia is a common finding among patients admitted to Internal Medicine Departments, but, although several studies have shown the influence of anemia on quality of life, mortality and morbidity in elderly patients, its relevance remains unclear in subjects who meet the criteria for patient with multiple comorbidities (MCP). Our objective was to analyze the prevalence and prognostic importance of anemia in a cohort of patients with multiple comorbidities. PATIENTS AND METHODS: a prospective study was conducted by consecutively selecting a cohort of patients who met criteria for MCP during a year and had anemia on admission. Analytical, sociodemographic, clinical and functional variables were collected at admission and one year later vital status, number of admissions and functional status were also assessed. The association of abovementioned variables with the presence of anemia and overall one-year mortality was analyzed by univariable and multivariable analysis. RESULTS: two hundred and thirty four patients were included, with a mean age (standard deviation) of 80.32 (9.2) years. Of them, 104 (44.5%) were females and 130 (55.5%) males and 60.59% had anemia. Leading causes of anemia were iron deficiency anemia (47.55%) and chronic illness anemia (29.37%). Overall one-year mortality rate was 55.94% among patients with anemia and 35.22% among patients without anemia (p = 0.003). After multivariable logistic regression analysis, variables independently and significantly associated with mortality were anemia, > 3 points on Charlson´s index and chronic respiratory failure. CONCLUSION: the prognostic impact of anemia reinforces the importance of a comprehensive evaluation of patients with multiple comorbidities.

Aliment Pharmacol Ther 2011; 34: 1159–1172 Summary Background Only a minority of alcoholics develop alcoholic liver disease (ALD) and allelic variants within genes encoding glutathione-S-transferases (GST) have been associated with ALD vulnerability with controversial results. Aim To assess the effects of GST polymorphisms on ALD by means of a genetic association study and meta-analysis. Methods We retrieved published studies on the relationship between allelic variants within GST genes and ALD by means of electronic database search. A meta-analysis was conducted in a fixed or random effects model. Calculations of odds ratios (OR) and their confidence intervals (CI), tests for heterogeneity of the results and sensitivity analysis, have been performed. A genetic association study comparing GSTM1, GSTT1 and GSTP1 genotype distribution among 279 alcoholics with or without ALD and 144 controls was also performed. Results Fifteen previous studies were identified analysing the association of ALD with polymorphisms within GST genes. After meta-analysis, we found a significant association between the possession of the GSTM1 null allele and the presence of ALD (OR = 1.43; 95% CI: 1.14, 1.78; P = 0.002) among alcoholic patients. A significant association was also found for the possession of the GSTP1 Val/Val genotype and the presence of ALD (OR = 2.04; 95% CI: 1.09, 3.80; P = 0.03). Conclusions Our results suggest that, among alcoholics, carriers of GSTM1 null genetic variant or Val/Val genotype of Ile/Val GSTP1 polymorphism have an increased risk to suffer from alcoholic liver disease. The role of glutathione-S-transferase as a potential therapeutic target in alcoholic liver disease is reinforced.

Supported by an Institutional Grant (FIS 99/1239) from the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovacion, and an Institutional Grant (MSC 2007/020) from the Plan Nacional sobre Drogas, Ministerio de Sanidad y Consumo, Madrid, Spain.

Background: The genetic basis for the predisposition to alcoholic liver cirrhosis (ALC) remains unknown. Increasing evidence supports a role for the nuclear factor (NF)-κB, the NF-κB inhibitor α (NFKBIA), and the peroxisome proliferator-activated receptor (PPAR)-γ in the pathogenesis of alcoholic liver disease, raising the possibility that common polymorphisms in genes encoding these molecules may confer susceptibility to ALC. The objective of this study was to analyze the relationship between common polymorphisms in NFKB1, NFKBIA, and PPARG2 genes and the presence of ALC. Methods: A total of 258 male alcoholics (161 without liver disease and 97 with ALC) and 101 healthy controls were genotyped for the −94ins/delATTG NFKB1, 3′-UTR+126G>A NFKBIA, and 34C>G PPARG2 polymorphisms. The association of these genetic variants with ALC was tested in alcoholic patients with alcohol abuse and alcohol dependence. A logistic regression analysis was further performed to analyze the model of inheritance. Results: We found an association between the presence of the deletion allele in NFKB1 polymorphism and ALC in patients with alcohol dependence. We found no association between NFKBIA and PPARG2 polymorphisms and the presence of ALC. Conclusions: The deletion allele of the −94ins/del NFKB1 polymorphism could be associated with a higher risk of developing ALC through an increase in inflammation, as supported by previous data.

Aims: To determine whether the functional polymorphism −592C>A of the interleukin (IL)-10 gene ( IL10 ) influences the development of alcoholic liver disease or alcoholism in alcoholic Spanish subjects. Methods: The −592C>A IL10 polymorphism was analyzed by the polymerase chain reaction and digestion with restriction enzymes in 257 male alcoholics [161 without alcoholic liver disease and 96 with alcoholic liver cirrhosis (ALC)] and 100 male healthy controls. Results: We found no association between the −592C>A IL10 polymorphism and ALC. Meta-analysis combining this result and data from previous studies failed also to show any significant association between this polymorphism and alcoholic liver disease. However, the frequency of allele A carriers (CA and AA genotypes) was significantly higher in alcoholic patients (defined as patients with abuse or dependence of alcohol) than in healthy controls. Conclusion: The −592C>A IL10 polymorphism is not related to the risk of ALC. Nevertheless, our study shows that alcoholism is associated with an excess of allele A carriers in alcoholic patients.

Alcohol consumption and hepatitis C virus (HCV) infection have a synergic hepatotoxic effect, and the coexistence of these factors increases the risk of advanced liver disease. The main mechanisms of this effect are increased viral replication and altered immune response, although genetic predisposition may also play an important role. Traditionally, HCV prevalence has been considered to be higher (up to 50%) in alcoholic patients than in the general population. However, the presence of advanced alcoholic liver disease (ALD) or intravenous drug use (IDU) may have confounded the results of previous studies, and the real prevalence of HCV infection in alcoholic patients without ALD or prior IDU has been shown to be lower. Due to the toxic combined effect of HCV and alcohol, patients with HCV infection should be screened for excessive ethanol intake. Patients starting treatment for HCV infection should be specifically advised to stop or reduce alcohol consumption because of its potential impact on treatment efficacy and adherence and may benefit from additional support during antiviral therapy. This recommendation might be extended to all currently recommended drugs for HCV treatment. Patients with alcohol dependence and HCV infection, can be treated with acamprosate, nalmefene, topiramate, and disulfiram, although baclofen is the only drug specifically tested for this purpose in patients with ALD and/or HCV infection.

It is well established that alcoholism is associated with imbalanced immune responses. To date, most relevant finding reported is the existence of an immunodepressed state which leads to a higher risk of suffering from severe infections in alcoholic patients. However, recent studies have shown that ethanol intake is followed by changes involving the synthesis and serum levels of specific cytokines as well as the activation of several different subsets of cytotoxic lymphocytes, that could be involved in the development of alcoholic liver disease. Accordingly, tumor necrosis factor-alpha plays a key role in the development of alcoholic liver damage through the induction of both apoptosis and necrosis of hepatocytes. This cytokine, together with interleukin (IL) 1, IL6 and several chemokines, facilitate the development of inflammation of the liver. Additionally, both transforming growth factor-beta and platelet-derived growth factor, act over stellate cells favouring hepatic fibrogenesis. The advances in the knowledge of the immunological mechanisms involved in alcoholic liver disease may lead to the discovery of new potential therapeutic targets, which may modify disease outcome in the near future.

In this paper, we comparatively analyze the effects of the following different stimuli on the production and intracellular accumulation of the interleukin (IL)-1 beta, IL-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), and IL-8 inflammatory cytokines in both normal human peripheral blood (PB) dendritic cell (DC) subsets and monocytes: lipopolysaccharide (LPS) versus Staphylococcus aureus cowan I (SAC) in the presence or absence of interferon-(IFN)-gamma-, cytokine secretion-blocking agents (brefeldin A alone versus brefeldin A plus monensin), and incubation periods (6, 12, and 24 h). For this purpose, a four-color multiple-staining direct immunofluorescence technique analyzed by flow cytometry was systematically used in all experiments (n = 19). Our results show that after stimulation, an important proportion of each of the two CD33(+) myeloid DC subsets as well as the monocytes produce significant amounts of all cytokines analyzed under each of the experimental conditions assayed. In contrast, CD33(-/+lo) lymphoplasmocytoid DC failed to produce detectable levels of any of the above-mentioned cytokines under the same stimulatory conditions. Upon comparing the different stimuli used, LPS was associated with higher percentages of cytokine-producing cells compared with SAC, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS and SAC. As regards the secretion-blocking agents, brefeldin A alone was superior to the combination of brefeldin A and monensin. This is because it was frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential analysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed among DC, which became even undetectable at 24 h, in the absence of a significant increase in cytokine secretion. In summary, our results show that from the experimental conditions assayed in this paper, to induce cytokine production by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-gamma) in the presence of brefeldin A alone.

Background: In the present study, we analyzed, at the intracellular level, the pattern of cytokine secretion by the major CD4 + and CD8 strong+ peripheral blood (PB) T-cell subsets in patients with chronic alcoholism, and we correlated it both with the ethanol (EtOH) intake status and with the presence or not of alcoholic liver disease. Methods: For that purpose, a total of 30 chronic alcoholic patients, 10 without liver disease (AWLD group) and 20 diagnosed with alcoholic liver cirrhosis (ALC) were studied. In all cases, flow cytometric measurement of intracellular expression of interferon-γ (IFN-γ), interleukin (IL)-2, and IL-4 was performed on PB CD4 + and CD8 strong+ T lymphocytes. Results: After studying AWLD patients, we found increased numbers of both CD4 + and CD8 strong+ PB T cells with detectable cytoplasmic levels of the IL-2 and IFN-γ T helper (Th)-1-associated cytokines, the greater increase being observed for this latter cytokine (p < 0.001 for CD4 + andp < 0.01 for CD8 strong+ T cells). Regarding ALC patients, the pattern of expression of intracellular cytokines by PB T cells was different depending on the status of EtOH intake at the moment of entering this study. Accordingly, as in AWLD patients, ALC individuals who were actively drinking also displayed increased numbers of both CD4 + and CD8 strong+ T cells expressing Th-1-associated cytokines. However, in these patients, expression of IFN-γ, although being significantly greater than that observed in control individuals (p < 0.05), was significantly lower than that in AWLD patients (p < 0.01 andp < 0.05, for CD4 + and CD8 strong+ T cells, respectively). After a withdrawal period of ≥1 yr, ALC patients did not show significant changes in the cytoplasmic expression of Th-1-associated cytokines compared with the control group; in contrast, these patients showed a marked increase on the proportion of CD4 + and CD8 strong+ T cells expressing IL-4, a Th-2-associated cytokine (p < 0.01). After considering the ratio between the number of T cells expressing Th-1 (IFN-γ)- and Th-2 (IL-4)-associated cytokines in each individual, we found that there was a significant imbalance in this ratio, with a predominance of IFN-γ-producing T cells over IL-4 + T lymphocytes during EtOH intake. Conclusions: Our results showed that in patients with chronic alcoholism, active EtOH intake is associated with a Th-1 pattern of cytokine production by PB T cells.

No previous studies have been reported on human alcoholism in which the pattern of cytokine secretion by natural killer (NK) cells is explored. The goal of the present study was to evaluate the role of NK cells in the production of cytokines in patients with chronic alcoholism, analyzing at the same time the possible relationship between cytokine production and both alcoholic liver disease and ethanol (EtOH) intake. A total of 30 chronic alcoholic patients-11 without liver disease [alcoholics without liver disease (AWLD) group] and 19 diagnosed of alcoholic liver cirrhosis-were included in this study. Twenty-five age- and sex-matched healthy volunteers were analyzed as controls. Production of interferon (IFN)-gamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-6 was performed on NK-enriched peripheral blood mononuclear cells (PBMC) after stimulation with IL-2 and IFN-alpha. In AWLD patients, the production of TNF-alpha was significantly reduced, compared with normal controls, under both IFN-alpha (p < 0.01) and IL-2 (p < 0.05) stimulation. In patients with cirrhosis, TNF-alpha production by PBMC enriched in NK cells varied depending on the EtOH intake status at the moment of evaluation. Accordingly, an increased concentration of this cytokine was detected in the supernatants of cirrhotic patients and active EtOH intake, particularly after IFN-alpha stimulation (p < 0.05); whereas, in patients with at least 1 year of alcohol withdrawal, TNF-alpha levels remained within normal range. The results on the production of IL-6 and IFN-gamma in AWLD and cirrhotic patients showed that only cirrhotic patients with a prolonged EtOH withdrawal period display abnormal production. Accordingly, in this group of patients, a significantly increased release of IL-6 was observed after both IFN-alpha and IL-2 stimulation (p < 0.01 and p < 0.05, respectively). By contrast, a lower IFN-gamma production (p < 0.005) was detected with respect to the control group. Our results point to the existence of an abnormal cytokine secretion by NK cells from chronic alcoholism patients, which depend on both the existence of liver disease and the status of EtOH intake.

Present information about the behavior of the different lymphoid subsets in alcoholic hepatitis (AH), including cells displaying cytotoxic activity, is scanty and contradictory. The aim of this study was to gain further insight into knowledge of the immunological abnormalities involved in AH and the possible role of ethanol (EtOH) consumption in these changes. We analyzed the distribution of a wide range of peripheral blood (PB) lymphoid subsets, both during active EtOH intake and after a 3-month withdrawal period, using multiple stainings with monoclonal antibodies and flow cytometry, as well as natural killer (NK) cytotoxic activity. AH patients entering the study were selected strictly; only those undergoing their first episode of AH with no other lesions at liver biopsy were enrolled. Regarding the alcohol intake period, the most striking finding was a significant increase of the absolute number of PB T cells affecting both CD4 + and CD8 + lymphocytes. These changes were associated with a higher expression of T-cell activation antigens, such as HLA DR and CDT11c. Simultaneously, a significant increase in both NK cells (CD3 - / CD56 + ) and the cytotoxic T cells coexpressing the CD3 and the CD56 molecules together with an increased NK cytotoxic activity were observed. By contrast, the CD19 + /CD5 + B-cell subset was significantly decreased. No significant changes were observed with EtOH withdrawal except in CD5 + B lymphocytes, which returned to normal values. Our results show that, in AH patients, a significant expansion of both activated T cells and NK lymphocytes occurs in the PB, which is associated with an increased NK cytotoxic activity. Interestingly these abnormalities persist during the withdrawal period.

Chronic alcohol intake is associated with an increased incidence of certain neoplasms. Natural killer (NK) cells have been considered to be involved in control tumor development and growth. The goal of the present study was to contribute to a better understanding of the effects of ethanol (EtOH) per se on the NK-cell population. Both patients with chronic alcoholism without liver disease (AWLD) and subjects with alcohol-induced cirrhosis (ALC) were carefully selected for this study. Immunophenotypical and functional studies of peripheral blood (PB) NK-cells were performed during active EtOH intake and after 3 months of a withdrawal period. In the AWLD group a significant increase in number of NK-cells (CD3-/CD56+) (P < .05) associated with a parallel increase in NK-cell lytic activity (P < .01) was observed. In addition, the number of cytotoxic T cells displaying the CD3+/CD56+ phenotype as well as CD8-/CD57+ NK-cell subset was also increased (P < .01 and P < .001, respectively). By contrast, in ALC patients with active EtOH intake (ALCET group), although a significant increase in the number of NK PB lymphocytes was observed (P < .05), NK lytic activity was depressed (P < .05), suggesting the existence of a decreased lytic activity/NK-cell. After 3 months of EtOH withdrawal, PB mononuclear cells (PBMC) from the AWLD group patients still displayed an increased NK cytolytic activity; in addition, the number of PB NK-cells (CD3-/CD56+ and CD8-/CD57+) and CD3+/CD56+ PB T cells continued to be increased. Independently of the duration of withdrawal period, in ALC patients EtOH withdrawal was followed by a slight decrease in the NK lytic activity of PBMC with respect to the values in active alcoholism phase; slight differences observed in the NK lytic activity in ALC patients who quit drinking could be related to the tendency to decrease of the number of NK-cells toward normal values. Furthermore, although an increase in NK cytotoxic activity after stimulation of PBMC with interleukin-2 (IL-2) was observed in ALC, it did not reach the levels observed in healthy subjects. Overall, our results show that the behavior of PB NK-cell population in chronic alcoholism is different according to both the moment of EtOH consumption and the existence or not of ALC. Alcohol by itself induced an increase in the number and lytic activity of NK-cells. By contrast, the NK cytolytic activity is constantly depressed in the stage of alcoholic cirrhosis, supporting the notion that immunosurveillance mechanisms may be affected in these patients.

Background:Polymorphisms in the microRNA (miRNA) regulatory pathways are novel functional genetic variants whose association with alcoholism susceptibility has not been previously studied. Given the potential relationship between certain miRNAs and alcohol use disorders (AUDs), this study was designed to explore the association between two polymorphisms within hsa-miR-146a and hsa-miR-196a2 genes and susceptibility to these diseases.Methods:Three hundred and one male patients with AUDs and 156 sex-matched healthy volunteers were enrolled. Polymorphisms were genotyped using TaqMan® PCR assays. Allele and genotype frequencies were compared between groups and logistic regression analysis was also performed to analyze the model of inheritance.Results:There was a significantly higher prevalence of allele C carriers (47.8%) of the miR-146a G>C polymorphism (rs2910164) among patients with AUDs when compared with controls (35.9%), and multivariable logistic regression analysis showed that the C allele was associated with these AUDs (OR = 1.615, 95% CI 1.067–2.442; P = 0.023). Neither the genotype nor the allele distribution of miR-196a2 polymorphism (rs11614913) was significantly different between groups.Conclusions:This is the first genetic association study to explore the relationship of miRNA polymorphisms with AUDs and to show an association of the miR-146a C>G rs2910164 allelic variant with this disease.

The aim of the present study was to analyze on chronic alcoholic patients the effect of ethanol (EtOH) withdrawal on the immune system through the investigation of the distribution of PB lymphoid subsets, using multiple-stainings with monoclonal antibodies and flow cytometry. For this purpose a group of 20 patients with active alcoholism without liver disease, negative for hepatitis virus, and without malnutrition was analyzed and followed for 9 months after alcohol consumption had been discontinued. Twenty-five age- and sex-matched healthy volunteers were included in the study. The following panel of monoclonal antibodies combinations (FITC/PE/PerCP or PE-Cy5) was used: TCR alpha beta/CD3/HLA DR, CD25/CD56/CD3, TCR gamma delta/CD3/HLA DR, CD45RA/CD45RO/CD4, CD3/CD8, CD19/CD5, and CD3/CD11c. Analysis was performed on at least 1,500 events/tube at flow cytometry using the Lysys II software program. During the alcohol intake period, the most striking findings were a significant (P < 0.05) expansion of the CD8+ T-lymphocyte subset, which coexpresses the activation associated antigens HLA DR and CD11c, as well as a significant increase in both NK-cells (CD3-/CD56+) and the T-cell subset with NK activity coexpressing CD3 and CD56 (P < 0.05 and P < 0.01, respectively). In addition, a decrease in the CD5+ B-cells (P < 0.05), associated with reduced serum gamma-globulin levels, was also observed. During alcohol withdrawal, a rapid decrease towards normal values of activated CD8+/HLA DR + and CD11c+ T-lymphocytes was observed as well as a normalization of CD19+/CD5+ B-cells and gamma-globulin serum levels; these changes might be directly related to EtOH suppression. Surprisingly, however, new immunological imbalances emerged in spite of the absence of alcohol intake. Thus, a progressive and significant expansion (P < 0.05) of CD4+ T-cells associated with an increased expression of the CD25 activation-related antigen and a preferential use of the CD45RO isoform by CD4+ T-cells were observed. In parallel, there was an even more evident increase (P < 0.01) in the number of PB NK-cells. Our results show that EtOH consumption induces changes in the immune system, its effects persisting or even becoming more evident after suppression of EtOH intake for a 9 month period.

We identified, in the transcriptome analysis of patients with alcoholic hepatitis (AH), osteopontin (OPN) as one of the most up-regulated genes. Here, we used a translational approach to investigate its pathogenic role. OPN hepatic gene expression was quantified in patients with AH and other liver diseases. OPN protein expression and processing were assessed by immmunohistochemistry, western blotting and enzyme-linked immunosorbent assay. OPN gene polymorphisms were evaluated in patients with alcoholic liver disease. The role of OPN was evaluated in OPN−/− mice with alcohol-induced liver injury. OPN biological actions were studied in human hepatic stellate cells (HSCs) and in precision-cut liver slices. Hepatic expression and serum levels of OPN were markedly increased in AH, compared to normal livers and other types of chronic liver diseases, and correlated with short-term survival. Serum levels of OPN also correlated with hepatic expression and disease severity. OPN was mainly expressed in areas with inflammation and fibrosis. Two proteases that process OPN (thrombin and matrix metalloproteinase 7) and cleaved OPN were increased in livers with AH. Patients with AH had a tendency of a lower frequency of the CC genotype of the +1239C single-nucleotide polymorphism of the OPN gene, compared to patients with alcohol abuse without liver disease. Importantly, OPN−/− mice were protected against alcohol-induced liver injury and showed decreased expression of inflammatory cytokines. Finally, OPN was induced by lipopolysaccharide and stimulated inflammatory actions in HSCs. Conclusion: Human and experimental data suggest a role for OPN in the pathogenesis of AH. Further studies should evaluate OPN as a potential therapeutic target. (Hepatology 2013;58:1742–1756)

Development of alcoholic hepatitis (AH) may be favored by the activation of the innate immune response. Recently, decreased numbers of circulating regulatory T cells (Tregs) have been reported in diseases associated with an immune activation status, but no studies have focused so far, in investigating the distribution of Tregs in chronic alcoholism and its potential association with liver disease. Here, we analyzed for the first time the frequency of peripheral blood (PB) Tregs and Treg subsets in AH and its relationship with the production of inflammatory cytokines by PB monocytes and dendritic cells (DCs).PB samples from 25 male patients with AH were studied; in parallel, 15 male chronic alcoholic patients without liver disease (AWLD) and 17 male healthy donors were also studied, as controls. The distribution of CD4⁺CD25hiCD127-/lo Tregs and their maturation subsets (naïve, central memory, and peripheral memory Tregs) was analyzed by flow cytometry. Spontaneous and in vitro-stimulated production of inflammatory cytokines by PB monocytes and DCs was analyzed by flow cytometry at the cytoplasmic level.Patients with AH showed decreased (p0.05) numbers of PB CD4⁺CD25hiCD127-/lo Tregs at the expense of all maturation-associated subsets, while AWLD and healthy subjects showed a similar (p0.05) distribution of PB CD4⁺CD25hiCD127-/lo Tregs. Interestingly, significantly increased amounts of spontaneously produced inflammatory cytokines were found among circulating monocyte-derived DCs and monocytes from AH (and AWLD) patients in comparison with healthy donors. Conversely, the ability of these cell subsets to produce cytokines after in vitro stimulation was lower (p0.05) in AH versus the 2 control groups.PB CD4⁺CD25hiCD127-/lo Tregs are significantly decreased in patients with AH when compared to both healthy and AWLD; this may contribute to explain the more pronounced activation of the innate immune response observed in AH, as reflected by an increased secretion of inflammatory cytokines by PB DCs and monocytes, and could facilitate the development of liver disease.

Previous studies have suggested that the effect of naltrexone in patients with alcohol dependence may be moderated by genetic factors. In particular, the possession of the G allele of the A118G polymorphism of the µ-opioid receptor gene (OPRM1) has been associated with a better response to naltrexone, although controversial results have been reported. The aim of this paper is to combine previous findings by means of a systematic review and a meta-analysis. We retrieved studies on the relationship between A118G polymorphism in OPRM1 gene and response to treatment with naltrexone in patients with alcohol dependence by means of electronic database search. A meta-analysis was conducted using a random-effects model. Calculations of odds ratio (OR) and their confidence intervals (CI) and tests for heterogeneity of the results have been performed. Six previous studies have analyzed the role of A118G polymorphism in response to naltrexone for alcohol dependence. After meta-analysis, we found that naltrexone-treated patients carrying the G allele had lower relapse rates than those who were homozygous for the A allele (OR: 2.02, 95% CI 1.26-3.22; P = 0.003). There were no differences in abstinence rates. Our results support the fact that the G allele of A118G polymorphism of OPRM1 moderates the effect of naltrexone in patients with alcohol dependence. This genetic marker may therefore identify a subgroup of individuals more likely to respond to this treatment.

The association between alcoholic liver disease (ALD) and tumor necrosis factor-a gene (TNFA) polymorphisms has been analyzed in several studies, but results have been conflicting. The main purpose of this study was to integrate previous findings and explore whether these polymorphisms are associated with susceptibility to ALD. The authors surveyed studies on the relation between TNFA gene polymorphisms and ALD by means of an electronic database search. A meta-analysis was conducted in a random-effects model. The association between ALD and the � 238G>A or � 308G>A polymorphism of the TNFA gene has been analyzed in 11 studies. Concerning the � 238G>A polymorphism, the authors found a significant association between possession of the A allele and risk of alcoholic liver cirrhosis (odds ratio ¼ 1.47, 95% confidence interval: 1.05, 2.07). Meta-analysis of the relation between the � 308G>A polymorphism and ALD did not show any significant association. Given the limited number of studies and the potential biases, more data are needed to confirm the association described for the � 238A allele. epidemiology; genetics; genome, human; liver cirrhosis, alcoholic; liver diseases, alcoholic; meta-analysis; review; tumor necrosis factor-alpha

Genetic factors may determine susceptibility to develop alcoholic liver cirrhosis, although it remains uncertain why only a minority of alcoholics suffers from this disease. A decrease in serum levels of interleukin-2 (IL-2) is usually found in alcoholic cirrhotics. In this study we examined the relationship between the -330TG IL-2 gene (IL2) polymorphism and alcoholic liver cirrhosis.Genotyping of the aforementioned polymorphism was done by polymerase chain reaction and digestion with restriction enzymes in 257 male alcoholics (161 without liver disease and 96 with alcoholic liver cirrhosis) and 101 healthy controls. A logistic regression analysis was performed to adjust for potential confounders and to analyze the model of inheritance.We found an association between the -330TG IL2 polymorphism and alcoholic liver cirrhosis: the frequency of the allele T carriers (genotype TT and GT) was significantly higher in alcoholics with cirrhosis (96.9%) than in those without liver disease (89.4%, P=0.043).We report for the first time that the possession of the -330T allele of the IL2 is associated with a higher risk of developing alcoholic liver cirrhosis and this fact may favor the progression of alcoholic liver disease.

Background: Preliminary data suggest that polymorphisms in cytokine genes may be involved in the genetic predisposition to alcoholic liver cirrhosis or alcohol use disorders. We thus analyze the association between these diseases and the following polymorphisms: −33T > C IL4, −174 G > C IL6, −251 T > A IL8 and 1188 A > C IL12B. Methods: 258 male alcoholics (161 without liver disease and 97 with liver cirrhosis) and 101 healthy controls were genotyped for the above mentioned polymorphisms. We examined the relationship between genotype and allele frequencies and the presence of disease, as well as the correlation with combinations of putative pro-inflammatory genotypes. Haplotypes were inferred using the expectation–maximization algorithm and haplotype frequencies were compared. Results: We found no statistically significant association between any of these polymorphisms or the combinations of pro-inflammatory polymorphisms and the risk of alcoholic liver cirrhosis or alcohol abuse or dependence. Haplotype analysis of the IL4 and IL12B polymorphisms did not show any statistical relationship either. Conclusions: Our results do not support the hypothesis that the analyzed polymorphisms confer differences in alcoholic liver cirrhosis or alcohol use disorders susceptibility.

Background Alcoholism is frequently associated with altered immune responses, limited information being available on its effects on dendritic cells (DC). In the present study we analyze the effects of chronic alcoholism on circulating DC. Methods For the first time we studied the numerical distribution of DC in peripheral blood (PB), their immunophenotype, and their ex vivo pattern of spontaneous cytokine secretion, in chronic alcoholic patients without liver disease (AWLD group; n=17) and active ethanol (EtOH) intake, as well as in subjects with alcohol liver cirrhosis (ALC group; n=21). Results A significantly decreased HLADR expression and an increased reactivity for CD123 was observed on PB DC from AWLD patients; additionally, increased secretion of interleukin (IL) 1beta, IL6, IL12, and tumor necrosis factor-alpha (TNFalpha) by DC was also noted in this group. Conversely, patients with ALC and at least 1 year of alcohol withdrawal (ALCAW group) showed a decreased number of total circulating DC, whereas ALC patients with active EtOH intake (ALCET group) had an abnormally low production of IL1beta and TNFalpha by PB DC. Conclusion Chronic alcoholism in the absence of liver disease is associated with an increased secretion of inflammatory cytokines by PB DC, whereas ALCAW and ALCET patients show decreased numbers of circulating DC and reduced secretion of these cytokines, respectively.

Background: Controversial results have been reported about the effects of alcoholism on the functionality of monocytes. In the present study we analyze the effects of chronic alcoholism on the intracellular production of inflammatory cytokines by peripheral blood (PB) monocytes. Methods: Spontaneous and in vitro-stimulated production of interleukin (IL) 1α (TNFα) by PB monocytes was analyzed at the single level by flow cytometry in chronic alcoholics without liver disease and active ethanol (EtOH) intake (AWLD group), as well as in patients with alcohol liver cirrhosis (ALC group), who were either actively drinking (ALCET group) or with alcohol withdrawal (ALCAW group). Results: A significantly increased spontaneous production of IL1β, IL6, IL12, and TNFα was observed on PB monocytes among AWLD individuals. Conversely, circulating monocytes form ALCET patients showed an abnormally low spontaneous and stimulated production of inflammatory cytokines. No significant changes were observed in ALCAW group as regards production of IL1β, IL6, IL12, and TNFα. Conclusion: Our results show an altered pattern of production of inflammatory cytokines in PB monocytes from chronic alcoholic patients, the exact abnormalities observed depending on both the status of EtOH intake and the existence of alcoholic liver disease. © 2007 Clinical Cytometry Society

It is well established that alcoholism is associated with imbalanced immune responses. To date, most relevant finding reported is the existence of an immunodepressed state which leads to a higher risk of suffering from severe infections in alcoholic patients. However, recent studies have shown that ethanol intake is followed by changes involving the synthesis and serum levels of specific cytokines as well as the activation of several different subsets of cytotoxic lymphocytes, that could be involved in the development of alcoholic liver disease. Accordingly, tumor necrosis factor-alpha plays a key role in the development of alcoholic liver damage through the induction of both apoptosis and necrosis of hepatocytes. This cytokine, together with interleukin (IL) 1, IL6 and several chemokines, facilitate the development of inflammation of the liver. Additionally, both transforming growth factor-beta and platelet-derived growth factor, act over stellate cells favouring hepatic fibrogenesis. The advances in the knowledge of the immunological mechanisms involved in alcoholic liver disease may lead to the discovery of new potential therapeutic targets, which may modify disease outcome in the near future.

Aims: In an attempt to explain differences in susceptibility to alcoholism and alcohol liver disease (ALD), different genes have been analysed, among them those encoding inflammatory cytokines. Thus, it has been reported recently that both the interleukin 1 receptor antagonist (IL1RN) and the IL1β (IL1B) genes may influence the risk of ALD in Japanese alcoholics. We analysed the distribution of single nucleotide polymorphisms (SNPs) located in the IL1A, IL1B, IL1R1 and IL1RN genes in alcoholic and non- alcoholic Spanish subjects. Methods: DNA samples were obtained from 139 male alcoholics, 78 of whom were diagnosed as alcohol dependent (32 patients with liver cirrhosis and 46 without ALD) and 61 as alcohol abusers (25 with liver cirrhosis and 36 without ALD). As a control, we studied 81 age- and sex-matched healthy volunteers. Results: Alleles �511 IL1B*1 and IL1RN*1 were represented more in alcoholic patients than in the control group. We did not find any association of alcoholism or ALD with polymorphisms in the IL1A and IL1R1 genes. Conclusions: We conclude that the proteins encoded by the IL1RN and IL1B genes may be involved in susceptibility to alcoholism in Spanish men, probably through a different pathway from that involved in the regulation of the inflammatory response.

SUMMARYDendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II+/lineage− were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16 ± 0.06% of the PB nucleated cells and 55.9 ± 11.9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12 ± 0.04% of PB nucleated cells and 44.53 ± 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor α-chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1β (97 ± 5% of positive cells), IL-6 (96 ± 1.1% of positive cells), IL-12 (81.5 ± 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-α) (84 ± 22.1% of positive cells) as well as chemokines such as IL-8 (99 ± 1% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production.