Alcohol,Vol. 11, No. 5, pp. 361-365, 1994 Copyright©1994ElsevierScienceLtd Primed in the USA. All rightsreserved
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Chronic Ethanol Consumption Prior to Retrovirus Infection Alters Cytokine Production by Thymocytes During Murine AIDS YUEJIAN
WANG*I" AND RONALD
R. W A T S O N t l
*Nutritional Sciences Program and ~fDepartment o f Family and Community Medicine, University o f Arizona, Tucson, A Z 85724 Received 19 N o v e m b e r 1993; A c c e p t e d 4 M a r c h 1994 WANG, Y. AND R. R. WATSON. Chronicethanolconsumptionprior to retrovirus infection alterscytokineproduction by thymocytes during murine AIDS. ALCOHOL 11(5) 361-365, 1994.-Chronic ethanol (EtOH) consumption may be a cofactor in the development of acquired immune deficiency syndrome (AIDS). As the thymus is an unique site for T cell maturation, we investigated whether thymocytes from EtOH consuming mice were more predisposed to aberrant cytokine production due to retrovirus infection. Adult female C57BL/6 mice were fed 4.5~e (v/v) in liquid diet or control liquid diet without EtOH for 10 weeks. All diets contained nutrients at only the recommended daily intake level for mice. Then all mice were infected LP-BM5 retrovirus and were fed control liquid diets without EtOH. The body and thymus weights were not affected by EtOH consumption. However, thymocyte number and proliferation, which had been reduced during routine AIDS, were significantly further reduced by EtOH use. The production of IL-2 and IL-6 by thymocytes, which was lessened during retrovirus infection, were significantly further suppressed by dietary EtOH at 6 weeks postinfection, whereas levels of IL-4 and IFN-~ by thymocytes, which were elevated during retrovirus infection, were significantly and slightly further increased by EtOH-treated mice prior to retrovirus infection, respectively. These data suggest that dietary EtOH consumption can modulate cytokine production by thymocytes, adversely affecting T cell differentiation, especially during retrovirus infection. These results provide additional evidence that EtOH consumption should be a cofactor during development of AIDS via producing altered cytokine production and then disrupting T cell differentiation. Ethanol
T cell differentiation
ACQUIRED immune deficiency syndrome (AIDS) is a clinical disorder caused by a human immunodeficiency virus (HIV), representing the end point in a progressive sequence of immunosuppressive changes. HIV-induced immunosuppression results in host defense defects, which render the body highly susceptible to opportunistic infections and neoplasm (2). The onset o f symptoms o f AIDS (low CD4 + Th cells count and opportunistic infections) is quite variable with a mean incubation time 3-10 years following seroconversion (13). The deleterious effects o f chronic EtOH consumption on immune response (15,20,26) could explain EtOH enhancing the host susceptibility to HIV infection and the subsequent development of AIDS (1,27). Thus, the frequent EtOH use seen often
in HIV-positive patients may act as a cofactor to speed the development of AIDS (21). Pro-thymocytes leave the bone marrow and arrive at the thymus where they find the appropriate environment, which let them complete their differentiation to become functional T cell. The generation of functional competent T cells from precursor populations within the thymus involves several stages of cellular proliferation and differentiation (11,18), which are mediated by cytokines (4). EtOH consumption has been shown to reduce number of thymocytes, thymus weight, thymocyte mitogenesis, and CD4 + and CD8 ÷ thymocytes (6,7,9). Consequently, the alteration in peripheral T cell subsets or T cell functions by EtOH consumption could be the
To whom requests for reprints should be addressed. 361
WANG AND WATSON consequence of changes at the thymus level including thymocyte cytokine production. EtOH-drinking mice prior to LP-BM5 retrovirus infection are more predisposed to develop peripheral immune dysfunction and aberrant cytokine production than normal mice with retrovirus infection (22). Thus, we proposed that the peripheral immune dysfunction or aberrant cytokine production in EtOH users prior to retrovirus infection during the development of murine AIDS may originate from damage of T cell maturation/differentiation in the thymus due to EtOH consumption. To test this hypothesis, chronic EtOH-fed mice were infected with LP-BM5 retrovirus infections causing murine AIDS, which functionally simulates human AIDS (24). In this study, thymocyte proliferation and its cytokine [interleukin-2, 4, 6 (IL), and IFN-~.] production were evaluated. MATERIALS AND METHODS
Animals and LP-BM5 Retrovirus Infection Female C57BL/6 mice, 5-weeks-old, were obtained from National Cancer Institute (Frederick, MD). Animals were cared for as required by the University of Arizona Committee on Animal Research. They were fed the AIN-76A synthetic chow diet (Dyets, Bethlehem, PA) ad lib, which contains only the recommended levels of nutrients. After 3 weeks housing of the mice in the animal facility, the mice randomly were assigned one of two groups. One group received normal liquid diet without EtOH as a control. Another group received liquid diet with EtOH (4.5070 v/v). After 10 weeks feeding, the retrovirus was administrated intraperitoneally to mice in 0.1 ml. The retrovirus titer was an ecotropic (XC) of 4.5 log10 PFU/ml, which induces disease with a time course comparable to that previously published (28). Infection of adult female C57BL/6 mice with LP-BM5 MuLV leads to the rapid induction of clinical symptoms with virtually no latent phase. Upon infection, all mice received the control liquid diet until sacrifice. The schedule of experiment is shown in Table 1.
EtOH Diet All mice were fed the National Research Council (NRC) liquid diet (29). Dietary ingredients were obtained from Dytes (#710279, Bethlehem, PA). All diets were made isocaloric by substituting 26070 of the total calories with ethanol in the ethanol diet and with dextrose in the control diet. In previous studies in our lab, we have found that higher dietary EtOH (e.g., more than 5070 v/v) significantly increased mortality of mice, and lower dietary EtOH (e.g., < 3070 v/v) did not cause significant change in immtme function. Therefore, the concentration of EtOH in a NRC liquid diet was 4.5070 (v/v) in this
TABLE 1 SCHEDULE OF DIETARY TREATMENT AND RETROVIRUS INFECTION
Dietary Treatment (4.5 % EtOH and Control Liquid Diet)
LP-BM5 Infection and all mice received normal liquid diet Without EtOH.
Sacrifice for Analysis
study. A pair-fed study was previously performed in our lab with no significant differences between the pair-fed group and ad lib group observed in diet consumption, body weight, the weight of thymus and spleen, immune functions, and cytokine production (29). Therefore, the diets were provided ad lib in this study. The average consumed volume of liquid diet was 12-15 ml per mouse per day for every diet. No significant difference in volumes of diet consumption between two groups was observed (data not shown).
Cytokine Standards and Antisera Rat anti-murine IL-2, IFN-'r, and IL-6 monoclonal antibodies and standard recombinant murine IL-2, IFN-3,, and IL-6 were obtained from Genzyme (Boston, MA). Rabbit antimurine IL-2 polyclonal antibody was obtained from Collaborative (Bedford, MA). Rat anti-murine IL-4, biotin rat antimurine IL-4 monoclonal antibodies, and recombinant murine IL-4 standard were obtained from PharMingen (San Diego, CA). Goat anti-murine IL-6 polyclonal antibody was obtained from R&D System (Minneapolis, MN). Rabbit anti-murine IFN-3, serum was prepared in our lab by immunizing rabbit with murine recombinant IFN-%
Preparation of Cytokine Production Thymuses were gently teased with forceps in culture medium (RPMI 1640 containing 10°70FCS, 2 mM glutamine, 100 units/ml penicillin and streptomycin, CM), producing a single cell suspension of thymocytes. 0.1 ml/well of thymocytes (1 × 107/ml) were cultured 96-well tissue culture plates (Falcon, Lincoln Park, N J) with CM. Cell concentration was counted and adjusted to 1 × 107 cells/ml. Thymocyte viability was more than 95070 as determined by trypan blue exclusion. The thymocytes were stimulated with Concanavalin A (ConA, 10/~g/ml, 0.1 ml/well, Sigma, St. Louis, MO, diluted in CM) for induction of IL-2, IL-4 and IL-6 with a 24-h incubation, and IFN-3, by a 72-h incubation at 37°C, 5070 CO2 incubator. After the incubation time indicated, the plates were centrifuged for 10 min at 800 × g. Supernatant fluids were collected and stored at -70°C until assayed by ELISA assay. No loss of activity has been noted during such storage.
ELISA for Cytokines All cytokines were determined by sandwich ELISA as described previously (23). Briefly, the wells of 96-well microtiter plates (Immulon II, Dynatech, Chantilly, VA) were coated overnight at 4"C with 50 #1 of anti-cytokine capture rat monoclonal antibodies specific for the measured cytokines, diluted to 1-4/~g/ml in 0.05 M bicarbonate buffer (pH 9.6). Then 50 /d of standard or sample cytokines diluted in culture medium were added for 2 h at 37"C. Then 50/zl of polyclonal and biotinylated monoclonai detecting antibodies diluted in PBS (1-4 tLg/ml) were added into each respective plate for another hour incubation at 37"C. Then 50 #1 of diluted strepavidinHRP (1 : 5000, Jackson, West Grove, PA), goat anti-rabbit IgG-HRP (1 : 5000, American, Qualex, La Mirada, CA) conjugates, and donkey anti-goat-lgG-HRP (1 : 5000, Jackson, West Grove, PA) were added to each respective well. The plates were then incubated as above for another hour, and washed four times with PBS-Tween once with PBS. Finally, 100/zl of substrate buffer of ABTS( Sigma, citrate buffer, 0.1 M, pH 4.2 containing 0.0307o H202) was added to each well, and the plates were allowed to develop for 20-30 minutes at room temperature. Optical density was determined at 405 nm
ETHANOL, THYMIC CYTOKINE, AND MURINE AIDS by Titertek Multiscan (Flow Lab, Mclean,VA). Sensitivity of the ELISAs for IL-2, IL-4, IL-6 and IFN--y were determined to be 156, 156, 320, and 78 pg per ml, respectively. No crossreactions between these measured cytokines in the ELISA kits were observed (data not shown).
Mitogenesis of Thymocytes Thymocyte proliferation was determined by thymidine incorporation as described previously (23). Briefly, thymocytes (1 x 107/ml) in 0.1 ml were cultured in 96-well flat-bottom cultured plates (Falcon) with ConA 0tg/ml) in CM at 37°C, 5% CO2 for 20 h. Then they were pulsed with 3H-thymidine (0.5 ~Ci/well,New England Nuclear, Boston, MA). After 4 hours, they were harvested by a cell sample harvester (Cambridge Technology, Cambridge, MA). Radioactivity was determined by a liquid scintillation counter (Tri-Carb, 2200CA, Packand, Lagunahills, CA). Data are presented as counts per minute (cpm).
Statistics All parameters were compared between two groups using two-tailed Student's t-test; p < 0.05 was considered a significant difference between two groups. RESULTS
Body and Thymus Weight As shown in Table 2, the body weight and thymus weight were not affected by dietary EtOH consumption.
Thymocyte Number and Proliferation Thymocyte numbers were slightly decreased by retrovirus infection compared to uninfected, normal mice. EtOH consumption prior to retrovirus infection significantly ( p < 0.05) further reduced thymocyte numbers compared to controls at 6 and 9 weeks postinfection. Thymocyte proliferation was decreased by murine retrovirus infection, and it was significantly (p < 0.05) further reduced by EtOH use compared to controls at 6 weeks postinfection, but not at 9 weeks postinfection (Table 3)
Production of Thymic Cytokines
TABLE 3 EFFECT OF ETOH CONSUMPTION PRIOR TO RETROVIRUS INFECTION ON NUMBER OF THYMOCYTES AND THYMOCYTE PROLIFERATION* Postinfection (Weeks) 6
Weight Postinfection (Weeks) 6 9
Groups Control EtOH Control EtOH
Body(gm) 28.7 29.1 30.6 31.6
± ± ± ±
2.3 4.2 5.0 2.5
59.8 65.6 66.7 68.5
± + ± ±
5.9 4.3 10.8 13.2
*Values are mean ± SD for 6-8 mice for each group. Body and thymus weight of uninfected, normal mouse (diet- and age-matched mice) are 30.2 ± 3.2 gm and 52.9 + 2.3 mg, respectively.
Number (x 107/thymus) 7.23 4.03 6.21 4.36
± ± ± ±
0.56 1.23t 1.63 1.17t
Proliferation (CPM x 1000) 13.3 9.0 15.8 17.6
± ± ± ±
3.8 2.It 2.8 6.2
*Values are mean ± SD for 6=8 mice for each group. The thymocyte number and proliferation of uninfected, normal mice (diet- and age-matched mice) are 8.05 ± 1.2 × 107 and 23.525 ± 1.453 CPM, respectively. tP < 0.05 compared to age-matched control. weeks postinfection and was enhanced at 9 weeks postinfection. EtOH consumption significantly ( p < 0.05) further reduced IL-2 production compared to controls at 6 weeks postinfection, but it had no effect on IL-2 secretion at 9 weeks postinfection (Table 4). IL-4 and IFN-,y levels produced by thymocytes were elevated during retrovirus infection. Increased IL-4 release by thymocytes during retrovirus infection was significantly (/7 < 0~05) further elevated by EtOH compared to controls at 6 and 9 weeks postinfection (Table 4). EtOHconsumption significantly ( p < 0.05) further reduced IL-6 production compared to controls at 6 weeks postinfection, but it had no effect on retrovirus-induced increased IL-6 secretion at 9 weeks postinfection (Table 5). Increased IFN-7 release by thymocytes during retrovirus infection was sightly (/7 > 0.05) further elevated by EtOH compared to controls at 6 and 9 weeks postinfection (Table 5). DISCUSSION Chronic EtOI-I consumption prior to retrovirus infection exacerbated thymic dysfunction and aberrant thymic cytokine secretion initiated by the retrovirus infection, even though EtOH use was discontinued for the 6 or 9 weeks after infection. Thus, chronic consumption of EtOH can act as a cofactor to aggravate immune dysfunction via affecting T cell differentiation, explaining the findings that EtOH users would TABLE 4 EFFECT OF ETOH CONSUMPTION PRIOR TO RETROVIRUS INFECTION ON PRODUCTION OF IL-2 AND IL-4 BY CONA-STIMULATEDTHYMOCYTES* Cytokines(ng/ml) Postinfection (Weeks) 6
Control EtOH Control EtOH
IL-2 production by thymocytes was decreased during mufine retrovirus infection at 6 postinfection compared to that from uninfected, normal mice and was elevated at 9 weeks post infection, whereas IL-6 production was suppressed at 6
TABLE 2 EFFECT OF ETOH CONSUMPTION PRIOR TO RETROVIRUS INFECTION ON BODY AND THYMUS WEIGHT*
Groups Control EtOH Control EtOH
IL-2 0.203 0.120 0.439 0.438
± ± ± ±
0.067 0.038t 0.030 0.025
IL-4 0.038 + 0.060 ± 0.114 ± 0.153 ±
0.015 0.013t 0.040
*Every sample was determined in triplicate, Values are mean ± SD for 6-8 mice for each group. Cytokine was secreted by 1 × 106 thymocytes in 0,2 ml. The concentrations of IL-2 and IL-4 produced by ConA-stimulated thymocytes from uninfected, normal mice (diet- and age-matched to experimental mice) are 0.354 ± 0.069 and 0.015 ± 0.009 ng/mi, respectively. tP < 0.05 compared to age-matched control.
WANG AND WATSON TABLE 5 EFFECT OF ETOH CONSUMPTION PRIOR TO RETROVIRUS INFECTION ON PRODUCTION OF IL-6 AND IFN-3, BY CONA-STIMULATEDTHYMOCYTES* Cytokines(ng/ml) Postinfection (Weeks) 6 9
Groups Control EtOH Control EtOH
IL-6 1.40 0.32 4.58 4.78
+ + ± ±
1.02 0.039t 1.54 1.04
IL-7 1.87 2.83 6.45 13.1
+ + + ±
0.098 2.38 2.00 9.60
*Every sample was determined in triplicate. Values are mean ± SD for 6-8 mice for each group. Cytokine was secreted by 1 x 106 thymocytes in 0.2 ml. The concentrations of IL-2 and IL-4 produced by ConA-stimulated thymocytes from uninfected, normal mice (diet- and age-matched to experimental mice) are 2.50 ± 0.42 and 0.560 ± 0.023 ng/ml, respectively. I"P < 0.05 compared to age-matched control. be more predisposed to accelerated immune dysfunctions due to murine retrovirus infection (22). These results provide additional evidence that EtOH consumption could be a cofactor during development of AIDS via producing altered cytokine production and then disrupting T cell differentiation. The EtOH consumption reduced the thymus weight (6,9). However, the results from this study indicated EtOH use prior to retrovirus infection did not reduce thymus weight thereafter. The major reason is thymus weight increases with lower number of thymocytes in retrovirus-infected mice (14). The histological studies showed the disappearance of defined areas (like the cortex and medulla) in thymuses during murine AIDS. Moreover, there are empty spaces between groups of cells that do not correspond to collagen fibers. Thus, the increase of thymus weight induced by retrovirus infection may offset the decrease induced by EtOH consumption. This evidence could explain EtOH consumption effect to reduce thymocyte number without change of thymus weight. Cytokines in the human and mouse thymus have been associated with an essential role in T cell development (4). In mice expressing a human 1L-2Rct chain transgene, thymocytes express a nonfunctional murine IL-2R/3-human IL-2Rct heterodimer resulting in the accumulation of T cell precursors in the thymus and periphery (4). Also, anti-IL-2Rt~ chain antibodies abrogate T cell development, which can be reversed by addition of IL-2 (30). Addition of IL-2 to intact lobes immersed in culture medium promotes the selective outgrowth of T cells expression ,ySTCR (5). Consequently, the inhibition of IL-2 production by EtOH use we observed may partly contribute to the changes in T cell subpopulations and differentiation in the thymus. IL-4 blocks T cell development by reducing the number of double-positive thymocytes (17). The constitutive production of IL-4 in IL-4 transgenic mice also results in the inhibition of double-positive thymocytes and mature periph-
eral T cell development (12,19). The increased level of IL-4 produced by thymocytes induced by retrovirus infection was further enhanced by EtOH consumption. Thus, we concluded that this increase may aggravate the impairment of T cell development in the murine AIDS, thereby causing secondary acquired T cell deficiency. IL-6 has been shown to promote the differentiation of Thy-I+IL-2R + donor thymocytes after intrathymic transfer into irradiated hosts (16). Thus, the suppression of IL-6 secretion induced by retrovirus infection at the beginning of infection may also contribute the abrogation of T cell development in the thymus. EtOH ingestion further decreased IL-6 production, thus EtOH should contribute to the disruption of T cell maturation. The physiological significance of increased level of IFN- 7 by thymocytes induced by retrovirus infection may be involved in the upregulation of MHC class I and II expression in the surface of thymic stromal cells, which play a crucial role in the positive/negative selection during T cell education (i.e., recognition "self" and "nonself") in the thymus. Thus, the dysregulation of IFN-'y release by thymocytes may reflect the failure of T cell education, leading to loss of tolerance to self antigens or reaction to nonself antigens. The tendency of increased IFN-~, levels in EtOH users prior to retrovirus infection would be expected to contribute the failure of T cell education. Thus, abnormalities of T cell maturation in the thymus may contribute the impairment of T cell response we and others observed in the peripheral tissues in animals or humans. The underlying mechanisms by which EtOH uses are more predisposed to disruption of T cell functions due to retrovirus infection is that EtOH-induced corticosteroid production (10). The thymus is very sensitive to corticosteroids, which cause apoptosis and loss of functions of thymocytes. In addition, EtOH interacts with the endogenous opioid system by: (a) production of certain EtOH metabolites including the isoquinolines that bind to opiate receptors, (b) modification of the binding properties of opiate receptors, and (c) alteration of the release, synthesis and posttranslational processing of endogenous opioid peptide (3,8). Thus, EtOH-induced changes in corticosteroid and opiate could he responsible for the changes in cytokine release by thymocytes, thereby altering T ceil maturation. In addition, EtOH consumption may facilitate the spread and replication of retrovirus infection via inhibiting immune response and increasing oxidative stress (25). In summary, the adverse effects of dietary EtOH consumption on T cell differentiation in the thymus via modulating cytokine secretion we observed during murine retrovirus infection may induce defect helper T cells, explaining aggravation of immune dysfunction by EtOH during murine AIDS by EtOH consumption (22). These findings support the notion that EtOH consumption could be a forceful cofactor in the development of AIDS. ACKNOWLEDGMENT This work was supported by NIHAA 08037.
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