EXECUTIVE SUMMARY

ORGAN-SPECIFIC RESEARCH

Cardiovascular/Cardiomyopathy
Program Portfolio
Endocrinology
Program Portfolio
Bone Disorders
Program Portfolio
Immunology and AIDS
Program Portfolio
Pancreatitis
Program Portfolio
Liver Injury
Program Portfolio

EMERGING CROSS-CUTTING ISSUES

Metabolism
Program Portfolio
Epithelial Cells
Cell-Cell Communication and Intracellular Signaling
Mechanisms of Disease and Disease Monitoring
Model Systems

REFERENCES

APPENDICES

A: Subcommittee for Review of Biomedical Research Portfolio
B: Experts in Biomedical Research
C: NIAAA Program Staff
D: NIAAA Staff and Guests

The National Institute on Alcohol Abuse and Alcoholism’s (NIAAA) Subcommittee for the Review of the Extramural Research Portfolio for Biomedical Research met on May 2-3, 2000. The charge to the Subcommittee was to examine the appropriateness of the breadth, coverage, and balance of the biomedical research portfolio, identifying research areas that are well covered and others which are either under-investigated or which otherwise warrant significantly increased attention. The Subcommittee was asked also to provide specific advice and guidance on the scope and direction of the Institute’s extramural research activities in the biomedical research area.

The Subcommittee for the Review of the Extramural Research Portfolio for Biomedical Research consisted of a chair, NIAAA Advisory Council member, and an advisory group of seven individuals. Five of these individuals have demonstrated expertise in alcohol-related areas, and four individuals have demonstrated expertise in non-alcohol-related areas (see Appendix A).

The review process was initiated by having experts (see Appendix B) in biomedical research prepare written assessments of the state of knowledge, gaps in knowledge, and research opportunities. NIAAA program staff (see Appendix C) presented the current extramural portfolio, categorized into the areas of cardiovascular, endocrinology, bone disorders, immunology, AIDS, pancreatitis, liver, metabolism, and training and career development. All information was shared with experts, selected NIAAA staff, and the chair and advisory group before the meeting.

A summary of FY 99 biomedical research awards is detailed below.

¹ includes SBIR awards and reimbursable funds.

On May 2-3, 2000, experts and NIAAA program staff made abbreviated presentations of their material followed by discussion among all of the participants, including representatives from other NIH Institutes and guests (see Appendix D). After completing this process, the chair and advisory group, with input from the experts, delineated the following list of research priorities.

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(1)   Determine the basis for tissue-specific differences in ethanol metabolism. This can best be accomplished by using transgenics (knockouts of various cytokines, ADH, etc.); examining the role of innate immune systems; examining metabolite patterns and actions; and differentiating between direct and downstream effects.

(2)   New models of ethanol injury need to be developed. Examination of existing transgenics and emphasis on cofactors (priming, sensitization) can facilitate this process.

(3) Biomedical effects of moderate ethanol consumption, including pattern of consumption, should be emphasized and requires the development of new models.

(4) Resources for recording and organizing results of microarray analyses should be made available.

(5) Additional epidemiological studies are required to determine the biomedical consequences of alcohol consumption, especially in the areas of alcohol metabolism and genetics. Utilization of trans-NIH initiatives will be helpful.

Additional gaps in knowledge and research opportunities were determined by experts in each of the areas covered and are listed in the text of the report.

ALCOHOL ACTIONS ON THE CARDIOVASCULAR SYSTEM
AND CARDIOMYOPATHY

State of Knowledge (Andrew P. Thomas, Ph.D.)

Alcoholism is now recognized as the most frequent identifiable cause of heart muscle disease, and it has been estimated that alcohol abuse may underlie as much as 50% of all cases of congestive cardiomyopathy (Regan, 1990). Moreover, subclinical depression of heart function is apparent in a large proportion of alcoholics (Rubin and Urbano-Marquez, 1994). Alcoholic cardiomyopathy reflects a low-output congestive heart failure, the clinical symptoms and pathology of which are indistinguishable from other forms of dilated cardiomyopathy (Regan, 1990). The disease progresses from a preclinical (or asymptomatic) stage, in which the contractile function of the heart is compromised, initially manifest as a decreased diastolic function. These initial deficiencies are compensated by a variety of mechanisms, including ventricular dilation and cardiac hypertrophy. Eventually the compensatory mechanisms breakdown and the disease progressed through cardiac decompensation to cardiac failure. Alcoholic cardiomyopathy usually takes ten years or more of excessive drinking to develop. Women are as sensitive as men to the cardiac effects of alcohol (Fernandez-Sola et al., 1997). Subclinical depression of function is also widespread in alcoholics (Regan, 1990). The depression of cardiac contractile function and the increase in heart mass is correlated with lifetime alcohol consumption, indicating that the toxic effects develop in a dose-dependent fashion (Fernandez-Sola et al., 1997). Reversal of alcoholic cardiomyopathy can occur with abstention from alcohol, even after the development of end-stage congestive heart failure (Guillo et al., 1997).

During acute exposure to alcohol in vivo, there are two opposing effects. Indirect stimulation of the heart can result from alcohol-induced elevation of catecholamines, whereas the direct effect of alcohol on the heart muscle is to depress contractility (Thomas et al., 1994). It has been shown that alcohol interferes with the cardiac action potential and depresses the calcium transients that underlie excitation-contraction coupling (Kojima et al., 1993). Since a direct depression of contractile function is an early hallmark of alcoholic heart disease, and compensatory mechanisms probably play a role in the further development of the disease, the acute effects of alcohol to interfere with cardiac excitation-contraction coupling may contribute to the initiation of the processes leading to cardiomyopathy. Elevated catecholamine levels may play a role in the hypertrophic response to alcohol (Adams and Hirst, 1990). Another potential site of alcohol action is in the generation of energy, with disruption of mitochondrial function (Cunningham and Spach, 1994).

Alcohol abuse is associated with increased risk for arrhythmia, and both acute alcohol intoxication and chronic alcohol consumption can lead to arrhythmias (Regan, 1990). Arrhythmias could result from effects of alcohol either at the level of the triggering mechanisms (altered action potential firing) or by creating the substrate through structural alterations in the cardiac tissue. The most common rhythm disturbances observed in connection with alcohol abuse are atrial fibrillation and ventricular dysrhythmia. In most cases, atrial fibrillation brought on by acute alcohol consumption reverses within 24 hours of cessation of alcohol intake (Zakhari, 1991). Several studies have reported prolongation of the QT interval as a result of alcohol, even in alcoholic patients without cardiac dysfunction (Rossinen et al., 1999), and this is a known risk factor for malignant ventricular arrhythmias. Such arrhythmias are one of the major factors precipitating sudden death in alcoholics (Regan, 1990). Electrocardiographic abnormalities in alcoholics are associated with an adverse prognosis, especially for sudden cardiac death (Day et al., 1993).

Potential mechanisms for alcohol-induced arrhythmias can be divided into disturbances in the initiation of the electrical depolarization, interference in the propagation of the electrical impulses, and alterations in the normal path and sequence of electrical excitation (Rosenqvist, 1998). Alcohol can also interfere with the regulation of heart rate and impulse conduction by neurohormones (Zakhari, 1991).

Effects of Alcohol on Coronary Artery Disease, Myocardial Infarction, and Atherosclerosis

A large number of epidemiology studies have suggested that moderate alcohol consumption is associated with reduced risk of coronary artery disease (CAD) (Goldberg et al., 1999). Nonfatal acute myocardial infarction has been reported to be more prevalent in abstainers than in drinkers consuming a broad range of alcohol, but when CAD mortality is considered, the protective effect of alcohol is restricted to light to moderate drinkers (Klatsky, 1994). This gives rise to a U- or J-shaped relationship between alcohol consumption and CAD mortality with a value of 1-2 drinks per day apparently affording optimal protection against CAD. It is clear from a wide variety of epidemiological studies that sustained consumption of high levels of alcohol (>3 drinks per day) is associated with increased risk of sudden death from other cardiovascular causes (Anderson et al., 1993).

It is likely that several mechanisms contribute to the apparent reduction in CAD risk in those consuming light to moderate alcohol amounts. Alcohol-induced increase in HDL can explain about half of the protective effect of alcohol against CAD (Langer et al., 1992). It has been noted that alcohol use is associated with elevated levels of the apolipoproteins associated with the formation of HDL, which are also closely correlated with reduced risk of CAD (Okamoto et al., 1988). Alcohol may also reduce CAD risk through an antithrombotic effect (Rubin, 1999). Important targets for alcohol inhibition in platelets include the thrombin-induced formation of the second messenger inositol trisphosphate and the release of arachidonic acid. Low levels of alcohol can enhance fibrinolysis, apparently by upregulating the expression of endothelial cell tissue plasminogen activator (t-PA) urokinase activity (Booyse et al., 1999).

In addition to the apparent protective effect of moderate alcohol consumption against CAD, moderate alcohol use may improve the recovery after acute myocardial infarction.

Chronic, excessive alcohol consumption is associated with hypertension, and this appears to be independent of other known risk factors for hypertension (Hillbom, 1998). In contrast to the effects of excessive consumption, a number of epidemiological studies have reported that moderate alcohol consumption is associated with a lower blood pressure than observed in abstainers (Hillbom, 1998).

Alcohol modifies the secretion of many hormones and neurotransmitters that regulate cardiac function and vasculature, which can result in changes in heart rate, force of contraction, vascular resistance, and the distribution of blood flow (Hillbom, 1998). Ethanol exposure increases sympathetic activity that can contribute to hypertension (Russ et al., 1991). It has also been reported that alcohol decreases the sensitivity of the baroreceptors, which provide feedback control over blood pressure by lowering heart rate and vascular resistance (el-Mas and Abdel-Rahman, 1993).

Alcohol can have direct effects on the contractile properties of vascular smooth muscle to affect blood pressure (Zakhari, 1991). Acute alcohol administration reduces intracellular calcium levels and can cause relaxation of aortic tissue (Zhang et al., 1992), whereas chronic alcohol exposure is associated with increased calcium fluxes into aortic smooth muscle (Vasdev et al., 1991). Of interest is the possible relationship between alterations in calcium fluxes and the hypomagnesmia observed in alcoholics (Altura et al., 1996).

Alcohol has been reported to have both positive (moderate drinking) and negative (heavier drinking) effects with respect to the risk of stroke. There is a clear association between heavy alcohol consumption and an increased incidence of stroke (Hillbom, 1999). It has been suggested that light alcohol consumption may have a protective effect against ischemic stroke (Gorelick and Kelly, 1992). A U- or J-shaped relationship was found between alcohol consumption and relative risk of stroke, with decreases in stroke incidence of up to 50% at alcohol consumption levels of 1-2 drinks per day. Nevertheless, higher levels of consumption are universally found to increase the risk of stroke (Anderson et al., 1993). The risk of stroke increased by 250-450% at consumption levels in the order of 5 or more drinks per day (Rodgers et al., 1993).

Several mechanisms are likely to contribute to the increased incidence of stroke in heavy drinkers. Other cardiovascular effects of alcohol described above are known risk factors for stroke. In particular, there is a well-characterized relationship between hypertension and enhanced risk of stroke. In addition, the effects of alcohol to interfere with blood clotting may contribute to the increased likelihood of hemorrhage. A further contributing factor may be alcohol-induced cerebrovasospasm, which is a local contraction of the muscle wall of blood vessels in the brain that can severely restrict or even block blood flow (Barbour et al., 1993). Altura and coworkers have suggested that the vasospasm induced by alcohol may result from a loss of cellular magnesium (Altura et al., 1993).

Specific recommendations:

(1) Determine the molecular changes that are associated with the development of alcoholic cardiovascular disease, using "gene-chip" array technology to screen for unknowns, targeted molecular approaches, and functional studies of specific components (particularly signal transduction, ion channels, and effectors of apoptosis).

(2) Determine the molecular and cellular mechanisms that underlie the protective effects of moderate alcohol consumption (including CAD, atherosclerosis, and recovery from acute myocardial infarction), focusing on expression and function of signal transduction pathways.

NIAAA PORTFOLIO ON CARDIOVASCULAR RESEARCH
(Thomas F. Kresina, Ph.D.)

Currently, the NIAAA Cardiovascular Research Portfolio supports 36 grants for a total of $5.5 million. Of these, 30 are basic research grants, five are career development awards and one award is a fellowship. (Table 1)

The NIAAA Cardiovascular Research Portfolio supports a wide range of research that includes both the apparent beneficial effects of moderate alcohol consumption and the toxic effects of chronic alcohol consumption on the cardiovascular system. As presented in Table 2, the scope of the portfolio comprises research addressing:

• The mechanisms of possible beneficial effects of moderate alcohol consumption on coronary artery disease and atherosclerosis
  
• The effects of moderate alcohol consumption on ischemia-reperfusion and myocardial infarction
  
• The effects of moderate alcohol consumption on stroke and thrombolysis

• The toxic effects of chronic alcohol consumption on the cardiovascular system

The NIAAA Cardiovascular Research Portfolio supports a balanced portfolio of research that includes both the possible beneficial effects of moderate alcohol consumption and the toxic effects of chronic alcohol consumption on the cardiovascular system. With regard to the mechanisms of possible beneficial effects of alcohol consumption, studies on coronary heart disease and atherosclerosis are investigating intracellular signaling pathways, lipid metabolism and adherence proteins. In ischemia-reperfusion /myocardial infarct studies, contractile function, Ca channels, cytoskeleton and cell surface receptors are targeted. In other studies, investigators are focused on fibrinolysis. Thus, the NIAAA has an active portfolio in all the areas of proposed cardioprotection: HDL cholesterol; platelet aggregation; reduced thromboxane synthesis; vasodilatation; LDL oxidation and free radical scavenging. In this area, recent studies have shown specific changes induced by alcohol in HDL constituents as well as in enzymes and transfer proteins that regulate HDL levels. Individuals who consume alcohol (1gm/kg/day) exhibit a significant increase in HDL-cholesterol levels.

In a study of chronic alcohol consumption and toxicity to the cardiovascular system, stress, hypertension, Ca signaling and cardiac function are being investigated. Recent studies have also shown that alterations in the concentration and composition of plasma lipids and lipoproteins arise with alcohol toxicity. With regard to ethanol-induced hypertension, tricuspid valve function has been shown to be impaired as well as mitral valve insufficiency due to alcohol consumption.

-  Further studies on mechanisms of possible beneficial effects of moderate alcohol consumption are needed. Once biological mechanism(s) are elucidated, then novel therapeutic approaches can be developed that provide cardioprotection.

-  Further studies are needed to elucidate the interaction of moderate drinking and cardiovascular medications

-  Understanding the mechanisms of alcohol-induced cardiomyopathy, hypertension and arrhythmia will lead to possible novel therapeutic interventions

Table 1. Grant Distribution by Funding Mechanism

Table 2. Number and Grant Support Level by Cardiovascular Category

Table 3. Grant Mechanism Distribution by Cardiovascular Category

State of Knowledge (Mary Ann Emanuele, M.D.)

In both males and females, the regulation of reproduction involves the hypothalamic-pituitary-gonadal (HPG) axis.

Acute or chronic ethanol ingestion suppresses HPG function resulting in, among other things, low serum testosterone, i.e., hypogonadism. While alcohol affects hypothalamic luteinizing hormone releasing hormone (LHRH) and pituitary luteinizing hormone (LH) in the adult (Cicero, 1982) and peripubertal male rat (Little et al., 1992), direct inhibition of testosterone steroidogenesis has been implicated as well (Johnston et al., 1981; Orpana et al., 1990). It is likely that ethanol is acting at more than one site of the testosterone synthetic pathway.

In testes, there are several potential, alcohol-influenced mechanisms for damage involving opioids (Emanuele et al., 1999), nitric oxide (Shi et al., 1998), the adrenergic system (Rivier, 1999), elevated pituitary prolactin and brain proinflammatory cytokines (Ogilvie et al., 1999), and perturbations in other hormonal systems that interact with the HPG axis.

The impact of ethanol exposure on hypothalamic LHRH in the male has been inconsistent and differs with the paradigm used. While secretion of LHRH has been reported to both be unaffected (Uddin et al., 1996) and reduced after ethanol (Ching et al., 1988; Hiney and Dees, 1991; Ogilvie and Rivier, 1997), the ability of the hypothalamus to synthesize LHRH appears to be unaltered by ethanol at any dose (Uddin et al., 1996).

Although there is a decrease in testosterone with ethanol exposure, the expected rise in serum LH does not occur, implying a central neuroendocrine effect (Emanuele et al., 1991). Studies in ethanol-fed rats have established that a decrease in LH blood levels results from impairment of both LH production and LH secretion. While there is less available data on FSH, the secretion of FSH does appear to be reduced by ethanol while FSH synthesis is unaffected (Emanuele et al., 1992).

Ethanol may increase opioids both directly and indirectly. While endogenous opioid peptides may mediate some of ethanol’s testosterone suppressive effects (Grattagliano et al., 1997), ethanol can also cause testicular oxidative injury and increase testicular apoptosis (Adams and Cicero, 1991).

Chronic ethanol exposure in the peripubertal age group decreases fecundity, which may be mediated by testicular oxidative injury leading to accelerated germ cell apoptosis in ethanol-exposed fathers (Little et al., 1992).

The major effect of chronic ethanol exposure in adult female rats is disruption of estrous regularity manifested mainly by a prolongation of diestrous (Eskay et al., 1981; Rettori et al., 1987). When proestrous occurs, it appears to be hormonally normal. Transient estradiol elevation (Lox et al., 1982), increase endogenous opioid peptides tone (Froehlich, 1993), and IGF-1 decline (Steiner et al., 1997) provide mechanistic bases for ethanol’s deleterious effects on female reproduction. In females, as in males, the onset of puberty is markedly disrupted by ethanol exposure, and one possible mechanism might be endogenous opioid peptides (Creighton-Taylor and Rudeen, 1991). Ethanol’s disruption of puberty may be in part owing to interference with the synthesis and secretion of IGF-I (Srivastava et al., 1995).

Acute ethanol exposure activates the HPA axis by inducing release of corticotropin-releasing factor (CRF) from the hypothalamus (Rivier, 1996). The HPA response to ethanol is dose-dependent, with activation at blood ethanol levels greater than 100 mg%, while at 75 mg% blunted ACTH and cortisol responses to exogenously administered CRF are noted, suggesting an attenuated ability of the HPA axis to respond to physiological stress (Wand, 1993). This impairment may result from ethanol-induced inhibition of arginine vasopressin, a secretogogue that potentiates the action of CRF on ACTH release (Wand and Schumann, 1998).

Chronic ethanol exposure is associated with increased as well as decreased HPA axis activity. The direct effects of chronic ethanol exposure on the HPA axis are difficult to assess due to concomitant problems, including malnutrition, depression, liver disease, and other stress factors. Individuals who are actively drinking and are non-depressed have been reported to have a two-fold increase in urinary cortisol levels and blunted ACTH and cortisol responses to CRF. These data support the contention that alcoholics have an abnormal HPA axis. In rodents, chronic ethanol exposure is not accompanied by elevations in ACTH and corticosterone, yet long-term influences in the HPA axis occur, including stress-related attenuation. Possible mechanisms include down-regulation of pituitary CRF receptors, increased corticosterone feedback, loss of responsiveness of nerve terminals, and hyperactivity of inhibitory neurons (Turnbull et al., 1999).

There is an increase in HPA axis activation during ethanol withdrawal; cortisol levels are increased as a result of elevation in cortisol burst amplitude and cortisol mass secreted per burst. Excess CRF, cortisol, and other neuroactive steroids (Devaud et al., 1996) enhance the magnitude of withdrawal symptoms, including seizure activity. Also disrupted is the normal circadian pattern of cortisol release and non-suppressability of the HPA axis to low doses of dexamethasone in chronic alcoholics during withdrawal. This hypercortisolism subsides over time with cortisol levels normalizing after 7-10 days of abstinence (Wand, 1993).

In early abstinence, hypo-responsiveness of the HPA axis is observed, with an attenuated hormonal responsiveness at each level of the axis. The attenuation of the HPA axis is still apparent after six months of abstinence.

A low-level cortisol response to ethanol has been shown to characterize individuals at high risk for alcohol abuse and dependence (Gianoulakis et al., 1996; Schuckit et al., 1988).

Chronic ethanol consumption is associated with increased prolactin release in men (Majumdar, 1979). Acute effects are more variable in women. Persistent hyperprolactinemia and associated menstrual cycle disturbances have been reported in heavily drinking women (Volpi et al., 1994). Inconsistent results on prolactin secretion have been observed in animals given ethanol. There is a marked inhibition of suckling-induced lactation in ethanol exposed post-partum female rats, associated with diminished pup growth (Subramanian et al., 1991)

Ethanol exposure is associated with suppression of the GH-IGF axis by lowering peripheral levels and altering the availability of these hormones (Srivastava et al., 1995), with younger animals being the most vulnerable (Steiner et al., 1997).

Acute and chronic ethanol abuse appears not to produce clinically relevant thyroid dysfunction (Garbutt et al., 1995). However, chronic alcoholics often display the nonspecific chemical features of the euthyroid sick syndrome, and resulting thyroid functions tests are often misleading, particularly in malnourished alcoholics with liver disease (Emanuele, 1997).

Although not a traditional neuroendocrine hormone, leptin has recently emerged as important to normal endocrine functioning, particularly in the reproductive arena. Ethanol may have alternating effects, acutely lowering leptin (Hiney et al., 1999) and chronically raising it (Lin et al., 1998). There may be a slightly different response in females since many studies have shown higher serum leptin levels in females than in males, a difference greater than can be accounted for by fat distribution alone.

Single bouts of ethanol exposure do not worsen and may improve glucose tolerance in diabetics. Some studies have indicated that isolated episodes of drinking with a meal may have a beneficial effect by slightly lowering blood glucose excursions (Swade, 1997). This potentially beneficial effect was observed in both men and women regardless of age. Studies of acute ethanol consumption in non-diabetic individuals have yielded variable results, with increases, decreases, or no change in glucose levels. However, daily drinking in moderate amounts (i.e., 0.5 to 1.0 mg/kg) clearly worsens diabetic control and increases the prevalence of impotence, retinopathy, and possibly peripheral neuropathy. The mechanisms underlying the hyperglycemia in chronically imbibing diabetics are still not fully known.

Specific recommendations:

(1) Study the role of oxidative injury and apoptosis as a common pathway in endocrine alteration.

(2) Examine differential sensitivity to humoral perturbations with age (adolescent to adulthood).

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NIAAA PORTFOLIO ON ENDOCRINOLOGY
(Thomas F. Kresina, Ph.D.)

Currently, the NIAAA Endocrinology Research Portfolio supports 31 grants for a total of $5.0 million. Of these 23 are basic research grants, two are career development awards and six awards are fellowships. (Table 1)

The NIAAA Endocrine Research Portfolio supports a wide range of research that includes the deleterious effects of alcohol consumption upon the endocrine system as well as energy regulation, metabolism, diet and oxidative stress. As presented in Table 2, the scope of the portfolio comprises:

• Research addressing the effects of alcohol on the hypothalamic-pituitary-adrenal (HPA) axis
  

• Research addressing the effects of alcohol upon the opioid system
  

• Research addressing the role of oxytocin on alcohol consumption
  

• Research addressing the effects of alcohol on the male and/or female reproductive system, the hypothalamic-pituitary-gonadal axis
  

• Research addressing endocrine-mediated immune suppression by alcohol
  

• Research on the effects of alcohol upon the endocrine mediators of osteogenesis and osteoporosis
  

• Research on the effects of alcohol and glucose transport
  

• Research on the stress axis comprising the HPA and sympatho-adrenal (SA) axes and alcohol
  

• Research on neuropeptides, energy regulation, lipid metabolism and alcohol

The NIAAA Endocrine Research Portfolio is predominantly comprised of R01 grants (Table 1) that support a broad range of research related to the integrated endocrine systems, including the HPA axis, the mesocorticolimbic system and the renin-angiotensin system (RAS). Alcohol's deleterious effects upon the endocrine system are pervasive. A variety of endocrine functions are affected, and the ensuing endocrinological imbalances range from metabolic and behavioral defects, to reproductive or immune dysfunction, to osteoporosis or development of cancer. Thus considerable effort is underway to identify the mechanisms by which alcohol disrupts particular components of the endocrine system, and to understand how the interrelationships between these pathways and their target organ systems are perturbed. The NIAAA has focused recent efforts on the neuroendocrine peptide regulation of alcohol consumption. The NIAAA sponsored a workshop on April 28, 1999 entitled "Neuropeptides and Alcohol Intake" which interfaced alcohol research, the opioids, the classical neurotransmitters and the newly described network of peptides and hormones that regulate food intake and energy metabolism. Although many actions of alcohol on neurotransmitters have been reported, the potential role of various peptides is a current area of research. Neuropeptide Y (NPY), a hormone that can function as a neurotransmitter in the brain, is known to stimulate appetitive behaviors. Recently, NIAAA-funded scientists reported that mice rendered NPY-deficient by elimination of the NPY gene consumed more alcohol than were NPY-intact mice. NPY-deficient mice also were less sensitive to the sedative effects of alcohol than controls. Both of these phenomena have been associated with increased risk for alcoholism in humans. Conversely, mice genetically altered to produce abnormally high levels of NPY showed a lower preference for alcohol and were more sensitive to alcohol’s sedative effects. These findings suggest that NPY is part of the neural circuitry involved in responses to alcohol.

Based on such data, a RFA was released in FY99 entitled "Peptide Regulation of Alcohol Intake" for support of grants in the current fiscal year. The Areas of Research of this RFA included the HPA axis, the opioid system, the RAS, food intake and energy homeostasis peptides and alcohol intake.

Expanding the focus nucleated by the alcohol/peptide RFA would be an appropriate future direction for the endocrine portfolio. A genetic approach to the study of peptides, the endocrine system and their action on alcohol intake would promote an ongoing institute initiative on the use of transgenic animals and knockout mice to further study alcohol addiction. Further initiatives can focus on endocrine dysfunction and alcohol consumption in the induction of disease pathogenesis such as obesity, diabetes or oncogenesis. This area of research is currently not addressed in the endocrine portfolio. Furthermore, the study of endocrine dysregulation due to alcohol consumption could be augmented by collaborative initiatives with the immunology/host defense portfolios where investigations involving interactions between endocrine and immune systems in the context of alcohol consumption can be pursued. For example, an initiative requesting research proposals on disease susceptibility to infectious pathogens could focus on alcohol/HPA/immune system interactions

Table 1. Grant Distribution by Funding Mechanism

Table 2. Number and Grant Support Level by Endocrine Category

Table 3. Grant Mechanism Distribution by Endocrine Category

State of Knowledge (Russell T. Turner, Ph.D.)

Alcohol consumption is generally considered a risk factor for osteoporosis based on the frequent finding of a low bone mass, decreased bone formation rate, and increased fracture incidence in alcoholics. Alcohol has also been shown to reduce bone formation in healthy humans and animals, and to decrease proliferation of cultured osteoblastic cells. On the other hand, it has been difficult to demonstrate alcohol-induced bone loss and increased fracture rate in population-based studies. Indeed, most studies have shown a positive association between alcohol and bone mass and no change or a decrease in fracture risk. Overall, the evidence generally supports a detrimental effect of chronic alcohol abuse on the skeleton of a sub-population of men and a neutral or generally beneficial effect of moderate alcohol consumption, especially in women. This latter putative beneficial effect may be due to a reduction in the age-related increase in bone remodeling associated with postmenopausal bone loss.

Specific recommendations:

(1)  Determine molecular mechanisms of action.

(2)  Develop interventions to prevent and/or reverse bone loss.

NIAAA PORTFOLIO ON OSTEOPOROSIS
(Vishnudutt Purohit, D.V.M., Ph.D.)

Osteoporosis afflicts about 20-25 million Americans and is linked to 1.5 million fractures every year. Women are more susceptible than men to this condition. National health care cost for osteoporotic women is estimated to be 5 billion dollars annually. Chronic heavy alcohol consumption is a risk factor for osteoporosis since it decreases bone mineral density, increases bone loss, and increases rate of bone fractures. On the other hand, moderate alcohol consumption appears to have beneficial effects as shown by increased bone mineral density in postmenopausal women. NIAAA-funded researchers are investigating the underlying mechanisms of the effects of moderate and heavy drinking on bones.

The National Institute on Alcohol Abuse and Alcoholism currently supports nine projects that investigate relationships between alcohol and osteoporosis. The portfolio consists of 7 R01s, 1 R03, and 1 R21 that can be divided into five broad categories: moderate alcohol–related, adolescent-related, pregnancy-related, cellular and molecular mechanisms, and stress-related studies (Table 1 and 2). In FY 1999, the total amount of funding for these projects was $1.3 million.

Moderate Alcohol Consumption-Related Projects: Two research projects focus on the relationship between moderate alcohol consumption and bone disorders. One project is investigating the effects of moderate alcohol intake on bone metabolism in women by measuring the levels of parathyroid hormone, collagen breakdown products, osteocalcin, vitamin D, and estrogen. The hypothesis being tested is that moderate alcohol consumption increases bone mineral density. The second project is evaluating relative risk/benefits of moderate alcohol consumption on osteoporosis. The hypothesis under consideration is that mineral content, density and biomechanical strength of bone will be greater, and biological markers and mediators of bone metabolism will be improved in monkeys consuming moderate amount of alcohol.

Animal Model of Osteoporosis: A rat model of osteoporosis is being developed to determine life long consequences of ethanol consumption on bone growth in the young, bone maintenance in middle age, and bone loss rate in aging animals. In addition, consequences of long term ethanol consumption on the severity and the rate of formation of ovariectomy-induced osteopenia are being investigated.

Adolescent-Related Project: Chronic ethanol consumption is known to suppress hypothalamic-pituitary-testicular axis that leads to decreased levels of plasma testosterone, an androgen required to maintain bone mineral density in males. In this project investigators hypothesize that suppression of reproductive axis by chronic ethanol in peripubertal period results in long term bone disorders including osteopenia.

Pregnancy-Related Project: This study investigates effects of alcohol on calcium metabolism in pregnant rats and their fetuses. Alcohol-induced perturbations in calcium metabolism during pregnancy may have significant implications for the development of osteoporosis.

Cellular and Molecular Mechanisms: Three active projects focus on the cellular and molecular mechanisms by which ethanol impairs bone cell proliferation and bone metabolism. Of these, one project is testing a hypothesis that ethanol inhibits osteoblast proliferation by interfering with a specific site in the tyrosine kinase phosphorylation pathway that leads to activation of Ras gene. The second project focuses on cellular and molecular mechanisms of alcohol’s effects on bone and mineral metabolism. A rat model is being developed to evaluate dose response for the long-term effects of alcohol on bone mass, bone cell number, and bone’s mechanical properties. In addition, alcohol’s effects on bone remodeling, recruitment of osteoblasts and osteoclasts, and expression of genes related to osteoblast-derived cell signaling peptides are being studied. Third project examines the hypothesis that alcohol alters bone metabolism indirectly by elevating the secretion of cytokines – IL-1 and TNF, which have been shown to be involved in postmenopausal osteoporosis. Both cytokines are known to mediate bone loss due to estrogen deficiency by increasing bone resorption and decreasing bone formation.

Stress-Related Project: This project examines the combined effects of chronic ethanol consumption and stress on bone quality, serum osteocalcin (a bone protein), and biosynthesis of enzymes of the catecholamine pathway. The results of this project may help to understand the mechanisms leading to osteoporosis in humans who are heavy alcohol consumers and subjected to environmental stressors.

1. Osteopenia caused by chronic alcohol administration in adolescent female rats is not completely reversible.

2. Chronic alcohol administration in adolescent, adult, and elderly female rats reduces bone density, and peak bone mass in both cortical and cancellous bones without affecting calcium regulating hormones. In adult female rats, chronic alcohol also decreased the amount of bone surface containing osteoblasts (bone forming cells) and wall thickness of tibia, a measure of osteoblast activity. These results confirm a direct deleterious effect of alcohol on bone forming cells.

3. Chronic alcohol is injurious to the adolescent male rat skeletal system and recovery is not complete after abstinence.

4. Impairment of osteoblastic phospholipase D signal transduction plays a critical role in mediating antiprolifertaive effect of ethanol on osteoblast.

5. As low as 3% of caloric intake of ethanol for four months impaired bone metabolism in adult female rats, and this effect was related to a decrease in the number of active osteoblasts.

Future Directions for Research

1. Examine the effects of moderate alcohol consumption on bone parameters in postmenopausal women with a consideration of a) patterns of drinking; b) types of alcoholic beverages; c) dietary supplements; and d) hormonal therapy. Are the reported beneficial effects mediated via hormones or growth factors?

2. Study the interactive effects of chronic ethanol intake and the hormones of hypothalamic-pituitary-gonadal axis on the skeletal system of human adolescents. In addition, investigate the mechanisms by which alcohol impairs bone formation in adolescent rats.

3. Investigate the interactive effects of growth hormone-IGF-1 axis and alcohol on bone remodeling process including effects on osteoblast proliferation and functions.

4. Identify the bone growth factors and bone cytokines that are directly affected by alcohol.

5. Identify signaling processes of osteoblast proliferation that are directly affected by alcohol.

6. Investigate the effect of alcohol on the functions of osteoclasts that are involved in bone resorption.

Table 1. Alcohol and Osteoporosis: Grant Distribution by Grant Mechanism

Table 2. Alcohol and Osteoporosis: Grant Distribution by Areas of Research

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CONSEQUENCES OF ALCOHOL CONSUMPTION ON
IMMUNE-ASSOCAITED DISEASES

State of Knowledge (Lynell W. Klassen, M.D.)

While the association of alcohol abuse with increased deaths from infections was made over 75 years ago, only in the past 15 years has serious investigative efforts been made to understand the role of alcoholic exposure on immune dysfunction. The more recent studies from both humans and animals suggest that alcohol produces significant alterations in immunoregulation. These abnormal immunoregulatory effects produce either immunodeficiency or autoimmune features and can lead to clinical disease. Complications associated with chronic alcohol abuse have a variety of other abnormalities, which can contribute to immune dysfunction, including malnutrition, vitamin deficiency, and advanced tissue damage such as liver cirrhosis.

The present knowledge of immune abnormalities that occur following significant alcohol exposure suggests the following clinical consequences.

· Decreased host defenses

- increased susceptibility to and progression of infections

· Decreased immunosurveillance

- increased cancer induction - increased tumor growth and metastasis

· Increased autoimmune reactions

- enhanced organ destruction by cellular mechanisms - presence of autoantibodies to normal
    proteins

· Induction of neoantigen

- organ specific dysfunction/damage

· Altered inflammatory responses

- abnormal cytokine activity - non-specific tissue damage and constitutional effects (fevers, weight
    loss)

Experimental and clinical studies have demonstrated that alcohol has multiple effects along the entire pathway of immune recognition, activation, differentiation, proliferation, and final effector activities.

Aldehyde-protein adducts derived from alcohol metabolism can induce an immune response to both the adducted epitope as well as to normal protein epitopes that have not undergone chemical alteration. Circulating antibodies to these adducts have been detected following chronic alcohol consumption. The detection of such adducts in the livers of alcohol-fed rats suggest that these immune reactive proteins may be important in producing an autoimmune-like reaction that can cause damage. There is a clear potential for cellular cytotoxic mechanisms to be directed against these protein adducts, thereby enhancing liver damage. Similar protein-aldehyde adducts have been detected in other tissues as a product of lipid peroxidation (Hill et al., 1998). The finding of MAA adducts in atherosclerotic coronary arteries raises the possibilities that alcohol-induced bioreactive proteins may accelerate a variety of other disease processes.

Alcohol has been clearly shown to modulate the ability of monocytes to process antigens and express immunogenic peptides to T helper cells (Szabo et al., 1993). In vivo studies suggest that this effect is mediated primarily by alcohol-induced reduction of IL-1b and an increase in TGF-b . Studies of delayed type hypersensitivity (DTH) have demonstrated that the effect of alcohol on APC activity is genetically linked, reversible, and is the main cause of DTH suppression (Waltenbaugh and Peterson, 1997). The clinical significance of these observations is that both quantitative and qualitative changes in an immune response can result in sub-optimal destruction of infectious agents with resultant clinical disease. Thus, any decrease in antigen processing efficiency can lead to increased bacterial or viral burdens and clinical infections.

Recent findings suggest that chronic exposure to excessive alcohol induces TH2 excesses with increased IL-10, decreased IL-12, and decreased INF-g production (Peterson et al., 1998). The consequences of this TH2 functional excess is a polyclonal increase in immunoglobulins following chronic alcohol ingestion, development of selective immunodeficiencies to specific infectious agents, blunted response to primary infections, and altered cytokine milieu causing abnormal inflammatory and fibrinogenic responses. While these findings suggest that chronic alcohol consumption results in increased TH2 function and potential dysregulation, its significance in human disease is still untested.

Acute and chronic alcohol exposure inhibits antigen-specific T cell proliferation (Szabo, 1999), but produces minimal effects on total B cell number (Romagnani, 1991). However, in vivo studies suggest that B cell differentiation is altered by alcohol, particularly by a decrease in IL-4 levels, which can inhibit both B cell proliferation and immunoglobulin class switching (Aldo-Benson et al., 1992). Chronic alcohol administration is associated with reduced natural killer (NK) cell numbers and activity (Cook et al., 1997).

Although many studies have correlated chronic or acute alcohol exposure with various cytokine changes, it is still unclear which isolated change may be biologically important. While in vitro studies suggest that alcohol has minimal effect on the ability of T cells to produce IL-2, in vivo studies suggest that chronic alcohol administration affects T cell utilization of IL-2, decreasing proliferation and ultimately down-regulating cell mediated responses (Jerrells et al., 1990). Human studies have documented a decrease in serum IFN-a , IFN-g , and IL-2 levels following alcohol exposure (Vicente-Gutierez et al., 1991). Acute alcohol administration has been shown to stimulate the production of prostaglandin E2, which also down regulates T cell proliferation and differentiation. Elevation of TNF-a following alcohol exposure has been described (McClain et al., 1993).

One of the most consistent immune abnormalities in alcoholics is a significant increase in serum immunoglobulin levels. It is often difficult to determine whether the elevated immunoglobulins are a consequence of chronic alcohol exposure or the result of subsequent liver disease. However, alcoholics without liver disease typically have elevations in IgA levels, while those with alcohol liver disease primarily have elevations of IgG. While the effect of alcohol in B cell function is probably minimal, a selective decreased antibody response is probably secondary to abnormal T cell function (Romagnani, 1991). Chronic alcohol exposure results in a generalized polyclonal activation of antibody production while at the same time there is often a decrease in specific antibody response following vaccinations. This pattern is also seen in classic autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. T cell function is diffusely decreased following high dose alcohol ingestion (Israel et al., 1986). Abnormal delayed type hypersensitivity has been demonstrated by a significant reduction in tuberculin and fungal skin tests, and T cell mitogenic stimulation is usually markedly reduced. Decreased cytotoxic T cell activity against viral infected cells has also been reported.

There is inadequate evidence to determine whether alcohol is a co-factor either in HIV susceptibility or in disease progression.

The consumption of alcohol has been linked with the clinical progression of chronic hepatitis C infection leading to progressive liver damage (Ostopowicz et al., 1998). Individuals who are both alcoholics and positive for hepatitis C infection have additive effects in the development of liver disease. However, it is unclear whether these are two independent processes, or whether both conditions may directly interact to produce progressive tissue damage.

Specific recommendations:

(1) Determine the mechanisms and significance of alcohol-induced TH1/TH2 polarization; including

- which intracellular signaling pathways are affected by alcohol and contribute to the TH1/TH2 change,

- which changes in cell surface receptors accompany alcohol exposure result in TH2 activation,

- what cytokine environmental milieu is inducted by alcohol to produce a TH2 effect,

- what modalities can reverse the TH1/TH2 phenotype (cytokines, cytokine inhibitions, anti-oxidants, etc.).

(2) Determine whether the development of immunoreactive products, e.g., aldehyde adducts, directly cause fibrinogenesis
     and liver damage and

- by which molecular mechanism,

- by which cytokines.

NIAAA PORTFOLIO ON IMMUNOLOGY AND HOST DEFENSE
(Leslie S. Isaki, Ph.D.)

Research Mechanisms

Descriptions of Projects Supported

• Majority (61%) of investigators examines the chronic effects of ethanol on host systems, whereas about 20% of researchers study the acute effects of ethanol. Another 20% of investigators study both chronic and acute effects of ethanol. Of this group, two projects examine the acute (or binge) effects within a chronic model of alcohol consumption in animals. (Table 3)
  

• Table 4 summarizes the grant distribution by organs targeted for study. These studies include both the direct and indirect effects of ethanol in promoting pathological conditions of the targeted organs and tissues.
  

• Clinical studies in human subjects are generally preclinical in nature and consist of isolation of peripheral blood cells and manipulation of these cells ex vivo.
  

• All grants in this portfolio focus on basic molecular mechanisms and include investigations into aspects of alteration of mediator profiles and function.

II. Summary of Research (Table 5)

Table 5 provides a summary of the scientific disciplines of the research projects within the Immunology portfolio. The table is divided first into two general areas: Innate and Adaptive defenses. Innate responses refer to non-immune host resistance which act as first-line defense whereas adaptive responses consist of acquired immunity involving antigen-specific lymphocytes.

Research grants examine the early phases of host defense mechanisms that are altered by ethanol exposure and include:

• Signaling changes in ethanol-induced phosphorylation of proteins that transduce signals for transcription and cell growth factors.
  

• Molecular targets of ethanol, which result in impairment of mechanical barriers of host, defense in the lung (surfactant and ciliary motility).
  

• Protein modification of ethanol-related alterations in the regulation and processing of proinflammatory cytokines and ribosomal proteins in hepatic mitochondria.

• The effects of ethanol on respiratory function following traumatic brain injury in a swine model.

Research projects investigate the ethanol-related impairments to mechanisms of host defense. The grants can be divided into two research classes: studies on cellular and molecular aspects of the function of the immune system and studies on the effects of alcohol exposure on the pathogenesis of infectious microorganisms.

• In both humans and mice, two types of CD4-bearing T helper lymphocytes (Th cells), termed Th1 and Th2, are defined both by the types of cytokine secreted and immune function. Alcohol-consumption is associated with impaired antigen-specific cell-mediated immunity, whereas antibody responses are unimpaired or enhanced in vivo after alcohol exposure. Grants that focus on ethanol-altered cellular immune processes examine the early events in antigen recognition, evaluate accessory cell function and mediator production, and elucidate the mechanism of polarization of the immune response toward Th2-driven humoral immunity and away from Th1-driven cell-mediated immunity.
  

• Identifying the cellular and molecular targets of ethanol are the goals of researchers who study infectious agents and alcohol exposure. Compromise of host immunity, by excessive alcohol consumption, is associated with impaired or altered mediator production and function. These dysregulated cytokine activities can result in altered recruitment of effector cells to sites of infection and diminished pathogen killing.
  

• Research grants on tissue and organ damage as a consequence of excessive alcohol consumption focus on mechanisms of ethanol-induced immunodeficiency and autoimmunity as primary factors that lead to tissue and organ injury. Similar to events in resolving infections, ethanol-induced injury is generally preceded by inflammation that is associated with parenchymal infiltration of leukocytes (neutrophils and lymphocytes) and increased release of reactive oxygen species.
  

• Investigators who examine the effects of alcohol exposure in utero on the developing immune system focus on both B and T cell lymphopoiesis.

Ethanol-induced impairments to host defense has progressed rapidly within the last decade. Although both cell-mediated and humoral (antibody) immune responses are affected by alcohol, the underlying mechanism(s) of alcohol's effects on immune responses are unknown. The emerging mechanisms are based primarily on dysregulation or disruption of cytokine interactions rather than on the direct effects of ethanol. Recent evidence indicates that the primary outcome of excessive alcohol consumption, whether it be infection or tissue injury, may have an etiological basis in exacerbation or prolonged inflammation. It is likely that there will be many targets that underlie the ethanol-induced alteration of host defense responses. Table 6 lists the research projects that target emerging areas of study.

Alterations in both innate and adaptive immune responses by alcohol are manifested in the increased frequency and severity of infections and in alcoholic liver injury seen in alcoholics. To better understand the effects of alcohol on the entire immune response, projects on innate host defenses and interactions of the innate and adaptive immune responses need to be significantly expanded. Innate immune recognition appears to be primarily mediated through Toll proteins which recognize conserved molecular patterns that are associated with microbial pathogens (such as lipopolysaccharide, LPS). Upon encounter with pathogens or after LPS stimulation, these pattern-recognition receptors activate intracellular signaling, most notably via the transcription factor NF-6 B, which results in the induction of a variety of effector genes. The role of Toll-like receptors in alcohol-induced tissue injury and immune system impairments are specific investigations that can be pursued to further understand the relationship between innate responses and initiation and progression of alcohol-related injury.

Trauma

Alcohol consumption is an integral causal factor in most types of traumatic injury. Not only does alcohol increase the frequency and severity of injury, but both the acute and chronic usage of alcohol impair the body’s normal physiological response to injury and significantly complicates medical management of the trauma patient. Basic science trauma research focussing on ethanol-impaired mechanisms that lead to dysregulation of cytokines, increase in immunosuppression, and exacerbation of complications arising from infectious agents, burns, and surgical procedures are important areas for the conduct of research.

Although the neurological and behavioral aspects of withdrawal are well studied, little is known about the immunology and host responsiveness to infectious agents and injury during alcohol withdrawal. Data from the research supported in this portfolio are mainly generated from either active drinking models (or during the period of ethanol exposure with in vitro models) or immediately after ethanol administration has been halted.

Scant data indicate that during the period of alcohol withdrawal, the host may still be vulnerable to the damaging effects of ethanol. Results from an alcohol binge model in animals suggest that elevated levels of oxygen-derived radicals detected during withdrawal may be a significant contributory factor to tissue injury. In a study of cardiovascular risk factors involving male alcoholics, cytokine profiles were altered compared to control subjects, and alcohol withdrawal induced potentially atherogenic changes in lipoprotein (a). Host responses during withdrawal have been observed for a period of several days to one year post-drinking, when differences in levels of proinflammatory cytokines were detected in groups of alcoholics with and without liver disease.

Table 1: Grant Mechanisms

* One active R01 grant in no-cost extension.
** One active R21 grant in no-cost extension.

Table 2. Grant Mechanisms of the Immunology (non-AIDS) Program

Table 3: Distribution by Duration of Alcohol Exposure