Early alteration in leukocyte populations and Th1/Th2 function in ethanol-consuming mice

BACKGROUND: Chronic alcohol consumption polarizes the immune response away from Th1-mediated cell-mediated immunity. In the present report we investigate the first onset of alteration in immune parameters during ethanol consumption in terms of changes in splenic leukocyte cellularity and surface phenotype as well as alterations in Th1 and Th2 function. METHODS: BALB/c and C57BL/6 mice were fed ethanol-containing liquid diets, were pair-fed an isocaloric liquid control diet, or were fed solid diet and water ad libitum for up to 12 days. At intervals during the feeding period, splenic leukocytes were assessed for phenotypic markers by flow cytometry and for their ability to support antigen-induced interferon-gamma (IFNgamma) production in a coculture system. Mice were bled at intervals throughout the feeding period, and serum immunoglobin E (IgE) and alcohol levels were determined. RESULTS: Data show that phenotypic and functional alterations occur within the first few days of alcohol consumption. Both liquid diets affect splenic cellularity, and by dietary day 5, ethanol-containing liquid diets further reduce B and NK cell numbers. The decline in B cells is accompanied by a concomitant decline in the amount of major histocompatibility complex class II expressed on this population. Functional alteration in Th1-mediated IFNgamma production occurred in the population fed ethanol-containing liquid diets by dietary day 5. Th2 function, as indicated by systemic serum IgE levels in these unimmunized mice, is increased by dietary day 6 to 8 and correlated with significant blood alcohol levels. CONCLUSIONS: Ethanol consumption by mice causes a rapid decrease in splenic cellularity accompanied by a decrease in Th1 function and a rapid increase in systemic IgE levels.     

Ethanol Ingestion Inhibits Cell-Mediated Immune Responses of Unprimed T-Cell Receptor Transgenic Mice

This paper introduces a transgenic ug) mouse in which the majority of the CWbearing T cells have T-cell receptors that react with ovalbumin (OVA) as a model for ethanol research. Although these Tg animals were bred onto the BALB/c genetic background, a strain generally considered to be nonpreferring in ethanol consumption, we determined that BALB/c mice would consume an ethanol-containing liquid diet, without significant mortality, and assessed alteration of specific immune responses. BALB/c, C57BU6 (B6), or (BALB/c x C57BL6)F1 hybrid (CB6F1) mice were fed LED containing 35,30,25, or 20% ethandaerived calories. Significant mortalii (>40%) was seen only in Wc and pronounced weight loss was seen in BALB/c, B6, CBEIFl mice when they were fed the diet containii the greatest ethanol concentration (LED35). Diets containing lesser amounts of ethanol did not cause mortalii. Liquid diets containing ≥30% ethand-derhred calories significantly impaired the chicken γ-globulbspecnic delayed hypersensitivity responses in Wc, B6, and CB6Flmice without significantly affecting the humoral immune response to sheep red Mood cells. We show that immunization of the Tg mice is not required for the development of a vigorous "debyed hypersensitivity" response to OVA or the I-A'-restricted peptide OVL_323–399 in mice fed standard solid lab chow or liquid control diet. In marked contrast, OVA Tg mice fed ethand show a profound inhibition of this immune response, indicating that ethand-induced inhibition of cell-mediated immunity occurs independently of antigen priming.     

Effect of chronic ethanol consumption on protective T-helper 1 and T-helper 2 immune responses against the parasites Leishmania major and Strongyloides stercoralis in mice

BACKGROUND: Chronic alcohol consumption has been associated with significant increases in the prevalence of infectious diseases, and it has been suggested that these increases are caused by a direct effect of ethanol on the immune response. The objective of this study was to determine whether chronic ethanol consumption would affect the development of protective immunity to Leishmania major, which is controlled by the T-helper 1 (Th1) subset of CD4 cells, and Strongyloides stercoralis, which is controlled by the Th2 subset. METHODS: Mice were fed ethanol-containing liquid diet (25% ethanol-derived calories), liquid isocaloric diet without ethanol, or solid chow and then exposed to either of the two parasites. The ability of the mice chronically consuming alcohol to eliminate the infections was determined, as were the levels of parasite-specific humoral and cellular immune responses. RESULTS: Mice chronically consuming alcohol were capable of eliminating both of these infections in a manner identical to the control mice. In addition, splenocytes from mice chronically consuming alcohol infected with L. major produced nitric oxide at the same levels as in control mice. Antibody responses were altered in a manner suggesting an increase in Th2 immunity and a decrease in Th1 immunity in the mice chronically consuming alcohol. In mice chronically consuming alcohol that were infected with S. stercoralis, eosinophils migrated to the parasite's microenvironment, and antibodies were produced at levels equivalent to those seen in control mice. CONCLUSIONS: Mice maintained on an ethanol-containing liquid diet had some alteration in their ability to produce Th1 and Th2 immune responses yet were capable of generating unimpaired protective Th1 and Th2 responses.     

Effect of Ethanol on Maternal and Offspring Characteristics: Comparison of Three Liquid Diet Formulations Fed during Gestation

Maternal blood alcohol levels, weight gain during pregnancy, parturition time, perinatal mortality, and postnatal growth of offspring were compared in groups of pregnant rats fed one of three ethanol-containing liquid diets (Kahn's formula = BSA diet, Revised Wiener's = RA6 diet, and Lieber-DeCarli's high protein 82C diet = LDA diet). The three ethanol diets all contained the same amount of ethanol-derived energy (36% of total energy), but differed in the amount of energy contributed by protein (17, 30, and 25%), fat (36, 24, and 13%), and carbohydrate (12, 10, and 27%), respectively. The experimental design also included dams that were pair-fed isocaloric ethanol-free versions of the three ethanol diets (designated BSP, RP6, and LDA, respectively) and a group of dams fed a pelleted casein-based solid diet (PC diet). All experimental diets were fed ad libitum from gestational day 7 to delivery. The effect of ethanol exposure in utero was most severe in mothers and offspring fed the BSA diet. The feed efficiency ratio (maternal weight gain/total dietary energy consumed) of this low-protein ethanol diet was less than that of RA6 or LDA diets. The feed efficiency ratio calculated for RA6 and LDA diets was not different from that of PC diet. Compared with rats fed RA6 and LDA diets, the rats that were fed BSA diet exhibited deficient maternal weight gain, greater parturition delay, impaired fetal growth, and increased perinatal mortality among the offspring. BSA dams had the highest blood ethanol levels of all groups fed ethanol diets, and exhibited the least difference in blood ethanol concentrations between the day (2 PM) and night (9 PM) periods of the diurnal cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Ethanol and Control Liquid Diets on the Hypothalamic-Pituitary-Thyroid Axis of Male Fischer-344 Rats

The effects of a high dextrose liquid diet containing ethanol and two different control liquid diets on serum and brain thyroid axis hormones and liver and brain deiodinase activities were studied in groups of adult male Fischer-344 (F-344) rats. Rats received either lab chow, ad libitum; a nutritionally complete 10% (w/v) ethanol liquid diet, ad libitum; a volume of either a high carbohydrate (HC) or a high fat (HF) isocaloric control liquid diet equal to the volume of diet consumed by rats given the ethanol diet; or the HC control diet, ad libitum. Consumption of liquid diets was measured daily and body weights recorded every other day throughout the study. Hormones were measured after 2, 4, or 8 weeks and deiodinase activities after 4 or 8 weeks. Also, groups of rats were given the 10% ethanol diet, ad libitum, or pair-fed the HC control diet intermittently for 8 weeks, and thyroid hormones and thyroid-stimulating hormone (TSH) response to thyrotropin-releasing hormone (TRH) were determined. Within 2 weeks rats became accustomed to all diets and thereafter weight gain was comparable in all groups. Small differences between serum thyroid hormones of rats fed the ethanol diet and pair-fed HC or HF controls may have been caused by lower T4 secretion in ethanol-fed rats. Marked differences in free and total T4 and T3 between F-344 rats fed liquid diets for 4 or 8 weeks and rats fed lab chow probably resulted from higher liver 5'-deiodinase activity in rats fed liquid diets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose- and Age-Dependent Effects of Prenatal Ethanol Exposure on Hippocampal Metabotropic-Glutamate Receptor-Stimulated Phosphoinositide Hydrolysis

Prenatal ethanol exposure reduces the density of the N-methyl-D-aspartate (NMDA) receptor agonist binding sites and decreases the capacity to elicit long-term potentiation (LTP) in hippocampal tormation of 45-day-old rat offspring. We hypothesized that prenatal ethanol exposure would reduce metabotropic-glutamate receptor (mGluR)-activated phosphoinositide hydrolysis also. Sprague-Dawley rat dams were fed a liquid diet containing either 3.35% (v/v) ethanol or 5.0% ethanol throughout gestation. Control groups were pair-fed either isocalorically matched 0% ethanol liquid diets or lab chow ad libitum. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulated inositol-1-phosphate (IP₁) accumulation via activation of the mGluR in offspring whose mothers consumed the 3.35% ethanol liquid diet was not different compared with the control groups. Furthermore, trans-ACPD stimulated IP₁ accumulation in 10 to 13-day-old offspring of the 5.0% ethanol diet group was not different compared with the control groups. However, trans-ACPD stimulated IP₁ accumulation was reduced significantly in 56- to 82-day-old offspring of dams fed the 5.0% ethanol liquid diet compared with the control groups. In contrast, bethanechol stimulated IP₁ accumulation, mediated via activation of muscarinic cholinergic receptors, was not affected by maternal consumption of either ethanol liquid diet. These results suggest both dose- and age-dependent effects of prenatal ethanol exposure on hippocampal responsiveness to trans-ACPD-activated phosphoinositide hydrolysis. Furthermore, the ability of the 3.35% ethanol diet to alter hippocampal NMDA receptors without altering the mGluR response suggests a differential sensitivity to the effects of ethanol exposure in utero among hippocampal glutamate receptor subtypes. Recent studies indicate that activation of mGluRs facilitates NMDA receptor-dependent LTP. Thus, higher blood ethanol concentrations achieved by consumption of the 5.0% ethanol liquid diet adversely affects an additional glutamate receptor mechanism associated with LTP. This additional effect may lead to an even greater impact of prenatal ethanol exposure on LTP than occurs when NMDA receptor function alone is affected by maternal consumption of more moderate quantities of ethanol.     

Effect of Postnatal Ethanol Exposure on Expression of Differentiation Antigens of Murine Splenic Lymphocytes

Ethanol is a recognized immunosuppressive agent in the chronic alcoholic. However, the effects of ethanol exposure on the developing immune system have not been extensively investigated. This study evaluated the effects of early postnatal ethanol exposure, via breast milk, on splenic lymphocyte differentiation antigen expression in offspring reared by ethanol-fed mice. Maternal mice were fed a liquid diet containing 20% ethanol-derived calories during pregnancy (E-P), pregnancy and lactation (E-PL), or lactation (E-L). Ad libitumfed (C) and pair-fed (PF) control groups, fed a control liquid diet, were included. Expression of differentiation antigens on splenic lymphocytes from 21-day-old offspring reared by females in 1 of the 3 ethanol exposure conditions was evaluated by flow cytometry. Offspring reared by E-P females had similar numbers of splenic lymphocytes as offspring reared by C and pair-fed during pregnancy (PF-P) females. In contrast, offspring reared by E-PL and E-L females had fewer splenic lymphocytes than both PF-PL and PF-L (respectively), and C offspring. The number of Thy 1.2⁺, CD4⁺, CD8⁺, and IgG⁺ (B-cell) splenic lymphocytes was reduced in E-PL and E-L offspring compared with PF and C offspring. E-P offspring had fewer CD4⁺ and IgG⁺ splenic lymphocytes than C, but not PF-P, offspring. The percentage of Thy 1.2⁺ splenic lymphocytes was significantly reduced among E-PL and E-L offspring compared with PF-PL and PF-L (respectively), and C offspring. These results suggest that ethanol exposure of female mice during pregnancy, pregnancy and lactation, or lactation alone, alters the phenotypic development of splenic lymphocytes of offspring reared by these females. The greatest effect on differentiation antigen expression occurred when females consumed ethanol during the period of lactation. We speculate that direct exposure of the nursing offspring to ethanol via the breast milk was responsible for the reductions in specific splenic lymphocyte populations. These data demonstrate that mice reared by females fed ethanol during the early postnatal period have a marked depletion of each of the major subpopulations of splenic lymphocytes, and that Thy 1.2⁺ lymphocytes are differentially sensitive to ethanol.     

Murine Model of Ethanol-Induced Immunosuppression

Alcohol abuse has been associated with an increased susceptibility to infectious diseases and certain tumors. On the basis of these observations, an effect of ethanol on the immune system has been suggested. We have used a mouse model system in which male C57Bl/6 mice were fed either Lieber-DeCarli liquid diet containing ethanol sufficient to supply 37% of the total calories or isocaloric control diet in a pair-feeding design to examine the effect of ethanol on the immune system. The group consuming the ethanol-containing diet maintained relatively stable levels of blood ethanol for the 8 days of feeding. Consumption of ethanol for 8 days resulted in a profound loss of thymus and spleen cells, and the recovery of thymus cellularity was delayed relative to the recovery of spleen cell numbers after ethanol feeding was discontinued. Proliferation of spleen lymphocytes to T-cell stimuli (concanavalin A and alloantigens) was diminished; however, B-cell proliferation to lipopolysaccharide was relatively unchanged in mice fed ethanol-containing diet. Also in ethanol-fed mice a significant decrease in the primary antibody response to sheep red blood cells but not to the T-independent antigen trinitrophenol-ficoll occurred. These data establish the murine model system as a means to define further the effect of ethanol on the immune system and host defense mechanisms.     

Effects of Levamisole on Ethanol-Induced Suppression of Lactational Immune Transfer in Rats

Maternal ethanol consumption in rats has been shown to inhibit lactational transfer of immunity to Trichinella spiralis (T. spiralis) from dams to their neonates. The purpose of this study was to determine if this depressed immune transfer could be altered by treating the dams with a known immunostimulatory drug during pregnancy and lactation. Groups of female rats were fed ethanol-containing or were pair-fed isocaloric control liquid diets for 30 days, infected orally with 1,000 T. spiralis larva, and then continued on diet for 10 days to allow the adult worms to establish. The animals were placed on chow diets (maximum 5 days) and mated 1 to 1 with males. On day 1 of pregnancy the animals were returned to their respective liquid diets through pregnancy and lactation. One-half of the ethanol-treated animals was given 15 mg/kg body weight of levamisole in the diet beginning on day 10 of pregnancy and continuing until day 17 of lactation. On day 19 of lactation, pups from all experimental groups were challenged orally with 200 T. spiralis larva, and killed at 3 or 8 days postchallenge. Assays for intestinal worm burdens, IgG anti-T. spiralis serum antibodies, and mesenteric lymph node cell proliferation were conducted. At both sacrifice periods, pups from ethanol-treated animals showed significantly higher intestinal worm counts (decreased immunity) and significantly lower titers of specific antibodies than the pups of pair-fed animals or pups of animals receiving levamisole in addition to ethanol. There were no differences between pups of the ethanol/levamisole dams and pair-fed dams in worm counts or antibody titers. No differences in mesenteric lymph node cell proliferation in response to T. spiralis antigen or to concanavalin A was observed between the three groups. These results indicate that administration of levamisole to ethanol-induced, immunosuppressed dams can reverse some of the deleterious effects normally seen in lactational immune transfer to suckling pups.     

The Effects of Ethanol Consumption on the Expression of Immunity to Trichinella spiralis in Rats

The effects of ethanol ingestion on immune responses of female rats against Trichinella spiralis (T. spiralis) infections were investigated. Female rats were pair-fed either ethanol-containing or isocaloric control liquid diets for 68 days, during which time they underwent one pregnancy cycle. For some studies, animals were fed ethanol diets for 68 days beginning prior to pregnancy and continuing through lactation and involution. They were infected on Day 7 of involution with 1000 L1 larvae of T. spiralis and tested for a primary rejection response 10 days later. To test for a secondary immune response, rats were infected with T. spiralis, placed on ethanol-containing liquid diet 15 days later, and after 68 days on diets, challenged with 1000 T. spiralis larvae and killed 3 days later. For primary immunized studies, ethanol-treated animals demonstrated significantly lower levels of anti-T. spiralis serum antibodies in ELISA, reduced rates of H3 thymidine incorporation by lymph node cells stimulated with T. spiralis antigen and significantly higher numbers of intestinal worm burdens (decreased immunity) compared with pair-fed controls. For animals sensitized to T. spiralis prior to pregnancy and given a secondary challenge during involution, no differences were found between ethanol and pair-fed animals in their ability to reject their worm burdens or in anti-T. spiralis serum antibody levels; however, ethanol-treated animals showed reduced rates of thymidine incorporation by lymph node cells when stimulated with T. spiralis antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Feeding Patterns of Rats Chronically Ingesting an Ethanol-Containing Liquid Diet

We compared the feeding patterns of rats ingesting a 36% ethanol-containing liquid diet for 30 days with those of rats pair-fed an isocaloric liquid control diet or provided control diet or ground rat chow ad libitum. Ethanol-fed rats consumed fewer calories per day and gained less body weight than rats fed control diets ad libitum. Daily caloric intakes were ∼50% lower during the first 10 days and 20% thereafter. Lower intakes in ethanol-fed rats occurred through a decrease in mean meal size rather than number of meals per day, although meals were more evenly distributed diurnally. Pair-fed rats ingested most of their food in one or two meals within a few hours of presentation. In a related experiment, a 4-hr duodenal infusion of ethanol at a rate comparable to that of ethanol ingestion resulted in plasma ethanol levels of 28 ± 4 mM and suppressed 5-hr intake by ∼40% by increasing the mean postmeal interval and satiety ratio. These results suggest that the suppressive effect of ethanol ingestion on food intake may be mediated in part by a post-gastric mechanism of ethanol action.     

The impact of alcohol on BCG-induced immunity against Mycobacterium tuberculosis

Background: Alcoholics are at heightened risk for developing active tuberculosis. This study evaluates chronic alcohol consumption in a murine model of vaccination with Mycobacterium bovis Bacille Calmette–Guèrin (BCG) and subsequent pulmonary infection with virulent Mycobacterium tuberculosis. Methods: BALB/c mice were administered the Lieber–DeCarli liquid ethanol diet or pair‐fed the liquid control diet for 3 weeks either before or after subcutaneous vaccination with M. bovis BCG. At least 3 weeks after BCG vaccination, groups of mice on the aforesaid diets were challenged with intratracheal infection with M. tuberculosis H37Rv. Lung mycobacterial burden, and lung and lung‐associated lymph node CD4⁺ lymphocyte production of tuberculosis‐specific interferon (IFN)‐γ were assayed. Popliteal lymph node lymphocytes from both dietary regimens undergoing BCG vaccination (in the absence of M. tuberculosis infection) were also evaluated for purified protein derivative–induced IFN‐γ production by ELISpot assay. Results: Mice begun on alcohol prior to vaccination with M. bovis BCG demonstrated impaired control of pulmonary challenge with virulent M. tuberculosis, as well as impaired lung CD4⁺ and popliteal lymph node T‐cell IFN‐γ responses. If BCG vaccination was delivered prior to initiation of alcohol feeding, the mice remained protected against a subsequent challenge with M. tuberculosis, and BCG‐induced immunity was not impaired in either the lung or the popliteal lymph nodes. Conclusions: Alcohol consumption blunts the development of the adaptive immune response to M. bovis BCG vaccination, which impairs the control of a secondary challenge with M. tuberculosis, but only if the alcohol exposure is begun prior to BCG vaccination. These results provide insight into mechanisms by which alcohol consumption impairs antimycobacterial immunity, including in response to vaccination and subsequent pathogenic challenge.     

Ethanol consumption modifies dendritic cell antigen presentation in mice

Background:  Alcohol consumption impairs type 1 cell‐mediated adaptive immune responses both in vivo and in vitro. The present study investigated the effect of alcohol consumption on antigen‐presenting cell (APC) populations and cytokine production. Methods:  BALB/c were fed ethanol‐containing, pair‐fed isocaloric liquid control, or solid diets for 11 days. Macrophage and dendritic cell (DC) populations were isolated by paramagenetic bead separation and used to present ovalbumin (OVA) to highly purified syngeneic CD4⁺ T cells derived from DO11.10 T cell receptor transgenic mice in coculture. DC isolated from diet‐fed mice were also used to present OVA to highly purified CD4⁺ T cells derived from antigen‐naïve DO11.10Rag2^?/? mice that are devoid of memory T cells. In vitro cytokine responses, interleukin (IL) ‐2, IL‐6, IL‐12, IL‐13, IL‐17A, and interferon‐γ (IFN‐γ) were measured by enzyme‐linked immunosorbent assay. Flow cytometry measured cell surface molecule expression. Results:  Alcohol consumption impairs delayed hypersensitivity responses (type 1) and enhances serum IgE levels (type 2). CD11c⁺ DC, but not F4/80⁺ macrophages, support cytokine responses by purified CD4⁺ T cells. CD11c⁺ DC derived from ethanol consuming BALB/c mice show diminished ability to support IFN‐γ responses by purified CD4⁺ T cells derived from DO11.10 or DO11.10Rag2^?/? mice. Subset analysis indicates that of the 3 “conventional” DC subsets found in mouse spleens, CD11c⁺CD8⁺ DCs are both responsible for OVA presentation and susceptible to the effects of ethanol. Ethanol consumption does not overtly alter the percent of splenic DC, but does increase the surface density of CD11c on these cells. Data show that cocultures containing purified CD4⁺ T DO11.10 cells and APC derived from alcohol‐consuming mice show decreased IL‐6, IL‐12, IL‐17A, and IFN‐γ and increased IL‐13 cytokine production in response to OVA stimulation. Conclusions:  Ethanol alters CD11c⁺CD8⁺ DC function, affecting cytokines responsible for adaptive immune responses. A unifying hypothesis for the underlying mechanism(s) of ethanol’s effect upon adaptive immune function is proposed.     

Choline fails to prevent liver fibrosis in ethanol-fed baboons but causes toxicity

To determine how choline supplementation affects the liver and whether it can protect against ethanol-induced liver injury, baboons were fed either normal or choline-supplemented diets, each with or without ethanol. Eighteen baboons were pair-fed for 3 to 4 years liquid diets with 50% of total energy as ethanol or isocaloric carbohydrate; ten animals were given our regular diets, whereas in eight the choline content was increased 5-fold. Six additional animals were fed individually with the control diets (with or without additional choline). With both ethanol-containing diets, ethanol intake was comparable and resulted in hepatic steatosis and striking mitochondrial lesions, with increases in serum bilirubin and SGOT, SGPT and glutamate dehydrogenase activities. In addition, of the five animals fed alcohol with the regular diet, one progressed to incomplete cirrhosis and two others developed perivenular and associated perisinusoidal fibrosis. Similarly, in the four baboons fed alcohol with choline supplementation, incomplete cirrhosis developed in one and perivenular fibrosis in two. Collagen deposition was demonstrated by immunoperoxidase with a specific antibody against procollagen Type III. These animals also displayed proliferation of myofibroblasts in the perivenular area and transformation of fat-storing cells to transitional cells in the perisinusoidal space, with associated enhanced collagen fiber deposition. Thus, in baboons, choline supplementation failed to prevent alcohol-induced steatosis and fibrosis. All parameters remained normal in the eight baboons fed the regular control diet. However, in the choline-supplemented controls, serum bilirubin, SGOT and glutamate dehydrogenase activities increased moderately and serum albumin decreased. Occasional fat droplets appeared in hepatocytes with mitochondrial changes (enlargement and alterations of the cristae) and an abundance of "myelin" figures in the cytoplasm, indicating that choline supplementation exerts moderate hepatotoxicity.     

Reactive free radical generation in vivo in heart and liver of ethanol-fed rats: correlation with radical formation in vitro.

Rats fed a high-fat ethanol-containing diet for 2 weeks were found to generate free radicals in liver and heart in vivo. The radicals are believed to be carbon-centered radicals, were detected by administering spin-trapping agents to the rats, and were characterized by electron paramagnetic resonance spectroscopy. The radicals in the liver were demonstrated to be localized in the endoplasmic reticulum. Rats fed ethanol in a low-fat diet showed significantly less free radical generation. Control animals given isocaloric diets without ethanol showed no evidence of free radicals in liver and heart. When liver microsomes prepared from rats fed the high-fat ethanol diet were incubated in a system containing ethanol, NADPH, and a spin-trapping agent, the generation of 1-hydroxyethyl radicals was observed. The latter was verified by using 13C-substituted ethanol. Microsomes from animals fed the high-fat ethanol-containing diet had higher levels of cytochrome P-450 than microsomes from rats fed the low-fat ethanol-containing diet. The results suggest that the consumption of ethanol results in the production of free radicals in rat liver and heart in vivo that appear to initiate lipid peroxidation.     

Prolonged Hya-Disparate Skin Graft Survival in Ethanol-Consuming Mice: Correlation With Impaired Delayed Hypersensitivity

BACKGROUND: Ethanol consumption impairs cell-mediated immunity and enhances humoral immunity. Among cell-mediated immune reactions, little is known of the effect of ethanol on chronic graft rejection. Allograft responses against the male-specific minor histocompatibility antigen, Hya, are widely used to study chronic graft rejection. METHODS: Female C57BL/6 (B6) mice were fed ethanol-containing liquid diets, were pair-fed an isocaloric liquid control diet, or were fed solid diet and water ad libitum. One week after diet initiation, the mice were grafted with split thickness, orthotopic male tail skin grafts, and the integrity of the grafts was monitored as the diet continued. Delayed hypersensitivity (DTH) was also determined in these same mice. In addition, Hya-cytolytic T-cell-deficient syngeneic major histocompatibility complex mutant B6.C-H2bm13 (bm13) and B6.C-H2bm14 (bm14) mice were assessed for skin graft rejection, DTH, and cytotoxic T-lymphocyte (CTL) activity. RESULTS: Ethanol-consuming female B6 mice are impaired in their ability to reject syngeneic male skin grafts and to develop Hya-specific DTH responses. To address the underlying mechanism, we show that Hya graft rejection correlates with DTH and not with CTL activity. Female B6 mice clearly differ from female bm13 and bm14 mice in their ability to generate CTLs against Hya antigen. Despite their inability to make Hya-specific CTL responses, bm13 and bm14 female mice, nevertheless, make Hya-specific DTH responses and ultimately reject Hya-disparate skin grafts, indicating that Hya-specific graft rejection results from DTH. Ethanol, by impairing Hya-specific DTH, inhibits Hya-specific skin graft rejection. CONCLUSIONS: We demonstrate that ethanol consumption impairs Hya-specific graft rejection. In addition, experiments with mice unable to generate anti-Hya CTLs support previous observations suggesting that DTH responses are sufficient to cause rejection of Hya-incompatible grafts.     

Effects of Maternal Alcohol Consumption on Milk and Blood Lymphocytes and IgG Antibody Levels from Trichinella spiralis-Infected Rats

Immunity to the intestinal parasite Trichinella spiralis can be transferred from the mother to the neonate during lactation. Previous studies in our laboratory showed that the passage of immunity to pups from ethanol-treated dams was depressed. This study examined the effect of ethanol consumption during pregnancy and lactation on the T. spiralis-associated immune components in milk and blood. Groups of female rats were fed either ethanol-containing or isocaloric liquid diets for 30 days before T. spiralis infection, mated and maintained on corresponding diets through pregnancy and lactation. Two-color flow cytometric analysis was performed for lymphocyte populations, enzyme-linked immunoabsorbent assay for specific IgG, and radial immunodiffusion assay for total IgG. The percentage of total T cells and their subsets, T helper cells and T cytotoxic/suppressor cells in milk and those in blood were similar between pair-fed and ethanol-treated animals. However, the percentage of natural killer cells in milk from ethanol animals was significantly reduced compared with the pair-fed group (33% vs. 54%). The percentage of activated or memory type T helper cell subset (OX22^?W3/25⁺) was significantly increased in the blood of the ethanol-treated group. Pair-fed animals showed higher T. spiralis-specific IgG antibody levels both in milk and blood compared with ethanol-treated animals. In ethanol-treated animals, specific IgG levels and total IgG concentration in milk were significantly lower than those in blood, whereas in pair-fed animals, only total IgG concentration in milk was lower than that in blood. This study indicates that ethanol consumption during pregnancy and lactation alters the maternal immune system. Decreased milk natural killer cells and depressed specific antibody levels in milk of ethanol-treated animals are possible contributing factors to the previously observed depressed lactational immune transfer to the pups.     

The Effects of Maternal Ethanol Consumption on Lactational Transfer of Immunity to Trichinella spiralis in Rats

Transient immunity to the intestinal parasite Trichinella spiralis can be transferred from the mother to the neonate during lactation. The goal of this study was to determine whether maternal ingestion of ethanol during pregnancy and lactation inhibited expression of anti- T. spiralis immunity in nursing pups. Groups of female rats were infected with 1000 T. spiralis L1 larva, mated, and fed either ethanol-containing or isocaloric liquid diets and maintained on diets through pregnancy and lactation or were fed the liquid diets for 30 days before T. spiralis infection, mated, and maintained on diets through pregnancy and lactation. Pups were challenged orally with 200 T. spiralis larva at 14 days postdelivery (preweaning period) or 21 days postdelivery (postweaning period) and were sacrificed either 3 or 8 days after respective challenge. Intestinal worm counts and serum titers of anti-T. spiralis IgG antibodies were determined for each pup. No difference in the number of intestinal worms between pups of ethanol-treated and pair-fed dams that received ethanol diet after T. spiralis infection was observed in the preweaning period. This was also true of pups from the dams sacrificed at 3 days after challenge in the postweaning period. However, similar pups sacrificed at 8 days after challenge showed significantly higher worm counts (decreased immunity) relative to their pair-fed controls. Pups of dams that received ethanol containing diet 30 days prior to T. spiralis-infection showed significantly higher numbers of intestinal worms relative to pair-fed pups at both the preweaning and postweaning periods.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-Buthionine (S,R) sulfoximine depletes hepatic glutathione but protects against ethanol-induced liver injury

BACKGROUND: L-Buthionine (S,R) sulfoximine (BSO) is an inhibitor of glutathione biosynthesis and has been used as an effective means of depleting glutathione from cells and tissues. Here we investigated whether treatment with BSO enhanced ethanol-induced liver injury in mice. METHODS: Female C57Bl/6 mice were pair fed with control and ethanol-containing liquid diets in which ethanol was 29.2% of total calories. During the final 7 days of pair feeding, groups of control-fed and ethanol-fed mice were given 0, 5 or 7.6 mM BSO in the liquid diets. RESULTS: Compared with controls, ethanol given alone decreased total liver glutathione. This effect was exacerbated in mice given ethanol with 7.6 mM BSO, causing a 72% decline in hepatic glutathione. While ethanol alone caused no decrease in mitochondrial glutathione, inclusion of 7.6 mM BSO caused a 2-fold decline compared with untreated controls. L-Buthionine (S,R) sulfoximine did not affect ethanol consumption, but serum ethanol levels in BSO-treated mice were nearly 6-fold lower than in mice given ethanol alone. The latter decline in serum ethanol was associated with a significant elevation in the specific activities of cytochrome P450 2E1 and alcohol dehydrogenase in livers of BSO-treated animals. Ethanol consumption caused a 3.5-fold elevation in serum alanine aminotransferase levels but the enzyme fell to control levels when BSO was included in the diet. L-Buthionine (S,R) sulfoximine administration also attenuated ethanol-induced steatosis, prevented the leakage of lysosomal cathepsins into the cytosol, and prevented the ethanol-elicited decline in proteasome activity. CONCLUSIONS: L-Buthionine (S,R) sulfoximine, administered with ethanol, significantly depleted hepatic glutathione, compared with controls. However, despite the decrease in hepatic antioxidant levels, liver injury by ethanol was alleviated, due, in part, to a BSO-elicited acceleration of ethanol metabolism.     

Increase of microsomal glucose-6-phosphatase activity after chronic ethanol administration

To study the effect of chronic ethanol administration on the activity of hepatic microsomal glucose-6-phosphatase, female rats were pair-fed liquid diets with 36% of total calories either as ethanol or isocaloric carbohydrate (controls). The remainder of the diet contained 35% of total calories as fat, 18% as protein, and 11% as additional carbohydrate. Six weeks of ethanol feeding as isocaloric substitution for carbohydrate increased significantly the activity of glucose-6-phosphatase (expressed per mg microsomal protein) both in fed (38%; p < 0.001) and fasted 18%; p < 0.02) rats. When expressed per unit of body weight, the enzyme activity was increased even further both in fed (66%; p < 0.01) and fasted (43%; p < 0.01) rats. Another group of rats received diets containing 36% of calories either as ethanol or isocaloric fat. The remainder of the diet contained 11% of total calories as carbohydrate, 18% as protein, and 35% as additional fat. Six weeks of this ethanol feeding as isocaloric substitution for fat again increased glucose-6-phosphatase activity significantly. Ultracentrifugation in a Cs⁺-containing sucrose gradient to separate rough and smooth microsomes revealed that the increase in glucose-6-phosphatase activity after ethanol feeding occurred mainly in the smooth microsomal membranes.