|NeuroAids Vol. 2, Issue 10 (November 1999)|
Interactions of Drugs of Abuse and HIV Dementia
A. Nath1,2, R.M. Booze3, K.F. Hauser3,
C.F. Mactutus4, J. Bell5, W.A. Cass3,
W. Maragos1,3, J.R. Berger1
5Department of Pathology (Neuropathology), Western General
Hospital, Edinburgh, Scotland, UK.
Although it has been recognized for several years that drug abuse is a significant risk factor for acquiring HIV infection, the magnitude of the problem has only recently been realized. As a result, the National Institutes of Drug Abuse (NIDA) is embarking on a major initiative to study this problem and to develop effective modes of intervention. NIDA director Dr. Alan I. Leshner recently remarked "Drug abuse and HIV are truly interlinked epidemics" (1). Where preventive measures fail, biological intervention prevails, hence it is imperatives that therapeutic measures be developed that intervene in counteracting the effects of drugs of abuse and HIV infection.
Drugs of abuse affect the central nervous system (2), and HIV infection is known to cause a dementing illness. Several studies have indicated the increased frequency of HIV dementia among the HIV-infected population that abuses intravenous drugs. Specifically, it was suggested that drug abuse potentiates the development of HIV dementia (3), perhaps by effects on the immune system. Italian investigators (4) have found a profound negative influence on cognitive function among HIV-infected persons. Pathological studies from a cohort in Scotland revealed that 56% of the brains of HIV-infected drug users had evidence of HIV encephalitis evidenced by the presence of HIV p24 and multinucleate giant cells, in comparison to only 15% of homosexual (non-drug users) HIV-infected men (5)(6). Others demonstrated that patients with a history of injection drug use who present with prominent psychomotor slowing have a more rapid neurologic progression and show abundant macrophage activation within the CNS (7).
Thus, neuroscientists have a key role to play in developing appropriate models for studying the mechanisms of these interactions and developing effective therapeutic measures for intervention. This article highlights some of the important questions that face neuroscientists today and invites discussion from other scientists on how best to address these issues. This article primarily discusses issues related to methamphetamine, cocaine and opiate use, although some aspects of the discussion are applicable to all drugs of abuse.
|Abstract||Prevalence||Effects||Mechanisms of Interaction||Experimental Systems||Conclusions||References|
|Drug abuse is a major cause of morbidity in young adults and has vast socioeconomic consequences for society. Combined with the fact that this population is also highly susceptible to contracting HIV infection, it has major health consequences. It is estimated that nearly 1.7 million Americans are cocaine users (half of whom use crack), 2.4 million report heroin use, and 4.9 million have used methamphetamine at sometime of their lives (1). Drug abusing populations initially ingest, smoke or inhale these drugs. But over a period of time they switch to intravenous drug use either due to development of tolerance or development of nasal ulcers. Injecting drug users are at higher risk for contracting HIV infection and of developing neurological and systemic complications. In nearly one-third of Americans infected with HIV, injection drug use is a risk factor making drug abuse the fastest growing vector for spread of HIV infection. Current estimates suggest that new HIV infections in injecting drug users outnumber those in homosexual male populations and at risk heterosexual populations (1). Also notable is that 70-80% of HIV infections attributable to heterosexual contact in the US have been in women. Most of these women are of childbearing age, with the greatest proportion reported to be minority women who are indigent and crack cocaine users and who trade sex for drugs and money (8). However, the spread of HIV among women is not limited to a particular ethnic or socioeconomic group, or particular intravenous drug. With increasing numbers of women acquiring HIV infection, basic research is needed aimed at understanding HIV infection in young women whose injection drug usage places them and their offspring at high risk. Thus, AIDS and drug abuse is a major woman's health issue with tremendous societal impact for current and future generations.|
|Effects on HIV Dementia|
Patients who engage in high-risk sexual behavior frequently abuse cocaine,
methamphetamine, opiates and alcohol (9). Pathological
studies also show that patients with HIV infection and a history of
drug abuse are more likely to develop HIV encephalitis (10).
Neurocognitive and motor dysfunction frequently occurs in the setting
of HIV infection; however, the degree to which drug abuse contributes
to these symptoms is largely unknown. Drug histories are frequently
not reliable, and patients are not routinely screened for drug use.
Experimental studies show that cocaine may contribute to the breakdown
of the blood brain barrier making the brain more susceptible to infection
with HIV (11). Methamphetamine and cocaine cause catecholamine
dysregulation. Patients with HIV infection also develop low levels of
catecholamines in their cerebrospinal fluid (12) and
clinical syndromes such as Parkinson 's disease (13),
and startle myoclonus (14) suggestive of dopaminergic
and noradrenergic dysfunction. However, the degree to which drugs of
abuse contribute to this clinical picture remains unknown. We have recently
reported a patient with a history of methamphetamine and cocaine abuse
combined with HIV infection. He developed a progressive dementia later
accompanied by bilateral focal dystonia, resting tremor, and choreoathetoid
movements, suggesting that these drugs of abuse may amplify the neurological
manifestations of HIV infection (15). Another study
using PET scans showed that cocaine causes decreases in cerebral blood
flow in the cortical regions of the frontal lobe even after the vasocontrictive
effects of the drug have reversed (16). It is thus
important to determine the degree of neuronal damage in patients with
HIV infection and drug abuse, and compare them to matched samples from
patients with HIV infection without drug abuse and to HIV negative patients.
Patients usually engage in abuse of multiple drugs, making it difficult
to determine how each drug contributes to the overall pathology. Animal
models are therefore necessary to systematically evaluate the effects
of neurotoxic HIV proteins in conjunction with methamphetamine, opiates
and cocaine on various cell populations in the basal ganglia, cortex
and hippocampus, and to identify cell populations that are vulnerable
to the combined toxicity of these substances.
Presymptomatic and Symptomatic HIV infection
|Mechanisms of Interaction|
Viral products released by HIV-infected cells cause widespread metabolic derangement, immune dysregulation, disruption of neuro-glial relationships, and neuronal dysfunction, all of which eventually cause cerebral dysfunction and contribute to the development of HIV dementia (17). Drugs commonly abused by HIV infected populations such as cocaine, methamphetamine and opioids all precipitate or augment varying degrees of neurotoxicity.
The mechanisms of interactions between HIV proteins and psycho-stimulants are not well understood. However, these compounds may interact at several levels. It has been shown that cocaine may cause a disruption of the blood brain barrier, hence increasing the ability of the virus and viral products to enter the brain (11)(18). The dopamine transporter may also be of particular importance in these interactions (19). Cocaine is a non-selective inhibitor of dopamine uptake by the dopamine transporter. Inhibition of the transporter results in a rapid increase of dopamine in the synaptic cleft, which has been hypothesized to underlie the euphoric "rush" that accompanies cocaine abuse. Cocaine has also been shown to affect cytokine production in splenocytes and macrophages but seems to have opposing effects in vivo and in vitro.
Amphetamines potentiate the release of dopamine into the synaptic cleft and may produce degeneration of dopamine terminals in humans who abuse them (20). Increasing dopamine levels in basal ganglia that are already impaired due to HIV infection may place the neurons at increased risk for toxic damage. Dopamine and metabolites can exhibit both excitotoxic and oxidative processes and may function in synergistic fashion to produce neuronal toxicity in the basal ganglia neurons in which dopamine uptake systems are present. gp120 blocks dopamine uptake in mesencephalic neurons in culture and results in loss of neuronal processes in dopaminergic cells (21). The effects of amphetamines on the immune system have not been well characterized.
Opiate drugs of abuse interact with HIV-1 at multiple levels in the pathogenesis of AIDS. Opiate drugs of abuse (such as heroin and morphine derived from the opium poppy) as well as endogenous opioids directly inhibit immune function (22)(23), which is likely to exacerbate the already devastating immunosuppressive consequences of HIV (23)(24)(25). In addition, opioids can potentiate HIV propagation in lymphocytes, microglial cells and monocytes (24)(25). A second level of interaction seemingly occurs between opiate drugs, HIV and non-immune target tissues. An important example where such an interaction is likely to occur is the nervous system, in which products of the HIV-1 gene, such as gp120 or Tat (17), may act synergistically with opiates to induce pathophysiological changes. Preliminary data from our laboratory suggests that morphine-induced activation of µ opioid receptors increase the toxicity of the HIV product Tat in striatal neurons (26) (unpublished observations). Importantly, neurotoxic interactions were noted in isolated neurons and astroglia in vitro, suggesting direct opioid-viral protein interactions in opioid receptor-expressing neurons and astroglia, which did not result from the effects of severe immunosuppression, secondary infection or abnormal cytokine signaling.
Moreover, the disruptive effect of opiate-HIV interactions may not be restricted to neurons. Based on findings that opiate drugs (27)(28) or viral proteins (17) similarly destabilize calcium homeostasis and increase reactive oxygen species in astroglia. We are conducting studies to determine if opioids and gp120/Tat would additively disrupt calcium homeostasis, increase oxidative stress in glia, and are currently testing this assumption (26) (unpublished observations). Similarly, methamphetamine and other drugs of abuse would also be expected to interact with HIV proteins to cause widespread neuroglial disruption. Hence, the nervous system may be a crucial site for deleterious drug abuse-HIV-protein interactions. Additional studies are needed to understand if drug abuse-HIV interactions cause either synergistic increases in oxidative stress, destabilize intracellular calcium, dysregulate cytokines or disrupt neuroglial function. These future studies would be enhanced by developing a clear understanding of the effects of the drugs of abuse on immune dysregulation within the nervous system.
|Abstract||Prevalence||Effects||Mechanisms of Interaction||Experimental Systems||Conclusions||References|
Use of well-characterized human tissues from patients with HIV infection who also abuse drugs will be essential in identifying mechanism for HIV-drug abuse interactions. Although these tissues are without a doubt necessary to define the extent of neuropathological damage in this patient population, difficulties associated with these studies include use of multiple drugs and therapeutic regimens and access to tissue only during terminal stages of the illness. For these reasons, substantial energies must be invested in developing in vivo and in vitro models best suited to study the interactions of HIV infection and drug abuse in the brain. However, given that humans are the only host for HIV infection, development of an ideal animal model is not possible. Therefore, careful consideration must be given to the inherent problems and advantages of each system.
Primates can be infected with related retroviruses such as simian immunodeficiency virus (SIV) and a chimeric strain of SIV and HIV. However, some important differences still exist in the neurological outcome of these animals when compared to humans. For example, the onset of symptoms is much more rapid in macaques and the encephalitis much more fulminant than is typically seen in humans (29). Further, sample sizes in primates must be limited due to the large amount of resources, expense and technical effort needed and the scarcity of these animals. The feline immunodeficiency (FIV) virus also causes encephalitis with neurodegeneration, although some differences exist with respect to the neuropathology seen in HIV encephalitis. Moreover, substantial differences exist between the FIV and HIV genome. However, this model may provide important information for certain targeted questions related to the effects of drug abuse in the setting of lentiviral infection and to monitor the effects of neuroprotective therapy (30).
An alternative strategy may be to use HIV virotoxins that have been implicated in the neuropathogenesis of HIV infection in conjunction with use well-characterized rodent models of drug abuse. The neurochemical and neurotoxic effects of cocaine, amphetamines, and opiates have been extensively examined in rats and mice. The neurotoxic properties of HIV proteins also have been studied in rodent nervous systems (31). The availability of a variety of mouse strains and/or the ability to generate transgenic mice, such as severe combined immunodeficiency mice (SCID), with altered responses to drugs of abuse or HIV are potentially compelling reasons for murine models to study particular drug abuse-HIV interactions in vivo or in vitro. Thus, the knowledge base for the effects of both drugs of abuse and HIV proteins in rodents provides a substantial background for examining interactive effects. Hence, rodents would serve as suitable in vivo models.
Additionally, cultures of human fetal brain cells would serve as an excellent in vitro model with neurotoxic properties of HIV proteins well studied in this system (17). Since adult human neurons cannot be grown in culture, fetal neurons can be differentiated in culture to express all glutamate and calcium channel receptor subtypes present in adulthood (32)(33). The proportions of glial cells can be manipulated in this system to address issues related to glial-neuronal interactions. Hence these cells represent an important model to address questions that require human brain cells. Again, because of the intricacy of drug abuse-HIV interactions, as well as ethical and practical concerns, no single approach or model is likely to elucidate this complex problem.
Rodent Models and Drug Pharmacokinetics
The general applicability of models for IV drug self-administration has been constrained by the use of chronic indwelling catheters that are exteriorized and tethered. A recent technical innovation is a SC implantable catheter as a port for the routine and repeated IV administration of drugs to group-housed rats (37). Using this implantable access port, cocaine-induced locomotor activity can be evaluated in freely moving rats following IV injections (38)(39). In our studies of adult male rats, IV administration of 3.0 mg/kg cocaine elevated arterial levels of cocaine by 30 seconds to approximately 2,500 ng/ml (34). This is comparable to the peak arterial levels associated with euphoric and cardiovascular effect in male human volunteers (35). Given that the risk factor for HIV infection in drug users is via needle sharing and injection drug use, the IV route of cocaine administration in rats is clearly preferable due to the similar route of administration and kinetic profile found in studies of at-risk drug abusers.
Drugs with a longer half-life produce more sustained plasma levels of the parent compound. Hence, the route of administration of an abused drug, such as methamphetamine, may not be as critical as that for cocaine in an animal model. However, peak arterial levels of the drug would nevertheless remain dependent upon the route of administration. Indeed, the rapid IV injection or smoking of methamphetamine may partially account for the growing preference of methods that allow drug abusers to control the rate of drug input (40). Although important differences in the neurotoxicity produced by methamphetamine may result as a function of administration route, this factor may be less critical in modeling repeated dosing. One pattern of human methamphetamine abuse is the administration of the drug multiple times during a run. A repeated dose protocol (4 injections/every 2 hours/one day) is sufficient to cause long term reduction in brain dopamine axonal markers (41).
Additionally, since humans frequently indulge in polydrug abuse, it is important to develop appropriate animal models using combinations of drugs (42). In particular, the simultaneous IV administration of cocaine plus heroin (speedball) may be of relevance to HIV seroconversion and neurotoxicity. Speedball injection is a risk factor for seroconversion (43). Speedball neurotoxicity may result from the synergistic effects of an indirect dopamine releaser (heroin) and a dopamine reuptake blocker (cocaine) via increasing dopamine levels (44). Together with HIV proteins present in the basal ganglia, speedball injections might further compromise the dopamine system.
|Abstract||Prevalence||Effects||Mechanisms of Interaction||Experimental Systems||Conclusions||References|
Drugs of abuse and HIV infection interact in the nervous system through a number of different mechanisms. Elucidation of these interactions requires complementary studies of human disease, animal models and culture systems.
Use the Back button in your browser to continue reading the article.
(2) Leshner AI, Koob GF (1999). Drugs of abuse and the brain. Proc Assoc Am Physicians 111(2):99-108. Medline
(3) Tyor WR, Middaugh LD (1999). Do alcohol and cocaine abuse alter the course of HIV-associated dementia complex? J Leukoc Biol 65(4):475-81. Medline
(4) Grassi MP, Perin C, Clerici F, Zocchetti C, Borella M, Cargnel A, Mangoni A (1997). Effects of HIV seropositivity and drug abuse on cognitive function. Eur Neurol 37(1):48-52. Medline
(5) Bell JE, Donaldson YK, Lowrie S, McKenzie CA, Elton RA, Chiswick A, Brettle RP, Ironside JW, Simmonds P (1996). Influence of risk group and zidovudine therapy on the development of HIV encephalitis and cognitive impairment in AIDS patients. AIDS 10(5):493-9. Medline
(6) Bell JE, Brettle RP, Chiswick A, Simmonds P (1998). HIV encephalitis, proviral load and dementia in drug users and homosexuals with AIDS. Effect of neocortical involvement. Brain 121 ( Pt 11):2043-52. Medline
(7) Bouwman FH, Skolasky RL, Hes D, Selnes OA, Glass JD, Nance-Sproson TE, Royal W, Dal Pan GJ, McArthur JC (1998). Variable progression of HIV-associated dementia. Neurology 50(6):1814-20. Medline
(8) Holmberg SD (1996). The estimated prevalence and incidence of HIV in 96 large US metropolitan areas. Am J Public Health 86(5):642-54. Medline
(9) Heckman TG, Kelly JA, Bogart LM, Kalichman SC, Rompa DJ (1999). HIV risk differences between African-American and white men who have sex with men. J Natl Med Assoc 91(2):92-100. Medline
.(10) Davies J, Everall IP, Weich S, McLaughlin J, Scaravilli F, Lantos PL (1997). HIV-associated brain pathology in the United Kingdom: an epidemiological study. AIDS 11, 1145-50. Medline
(11) Fiala M, Gan XH, Zhang L, House SD, Newton T, Graves MC, Shapshak P, Stins M, Kim KS, Witte M, Chang SL (1998). Cocaine enhances monocyte migration across the blood-brain barrier. Cocaine's connection to AIDS dementia and vasculitis? Adv Exp Med Biol 437, 199-205. Medline
(12) Berger JR, Nath A (1997). HIV dementia and the basal ganglia. Intervirology 40, 122-31. Medline
(13) Mirsattari SM, Power C, Nath A (1998). Parkinsonism with HIV infection. Mov Disord 13, 684-9. Medline
(14) Maher J, Choudhri S, Halliday W, Power C, Nath A (1997). AIDS dementia complex with generalized myoclonus. Mov Disord 12, 593-7. Medline
(15) Nath A, Maragos W, Avison M, Booze RM, Schmitt F, Anderson C, Berger JR. Seizures, focal dystonia, choreoathetosis and resting tremor with HIV dementia, methamphetamine and cocaine use: Synergistic neurotoxicity. Submitted.
(16) Bell KM, Milne N, Lyons KP (1994). Regional blood flow and cocaine abuse. West J Med 161:412-413. Medline
(17) Nath A, Geiger JD (1998). Neurobiological Aspects of HIV infections: neurotoxic mechanisms. Prog Neurobiol 54, 19-33. Medline
(18) Zhang L, Looney D, Taub D, Chang SL, Way D, Witte MH, Graves MC, Fiala M (1998). Cocaine opens the blood-brain barrier to HIV-1 invasion. J Neurovirol 4, 619-26. Medline
(19) Scarponi M, Bernardi G, Mercuri NB (1999). Electrophysiological evidence for a reciprocal interaction between amphetamine and cocaine-related drugs on rat midbrain dopaminergic neurons. Eur J Neurosci 11, 593-8. Medline
(20) McCann UD, Wong DF, Yokoi F, Villemagne V,
Dannals RF, Ricaurte GA (1998). Reduced striatal dopamine transporter
density in abstinent methamphetamine and methcathinone users: evidence
from positron emission tomography studies with [11C]WIN-35,428.
J Neurosci 18, 8417-842. Medline
(21) Bennett BA, Rusyniak DE, Hollingsworth CK (1995). HIV-1 gp120-induced neurotoxicity to midbrain dopamine cultures. Brain Res 705, 168-76. Medline
(22) Donahoe RM, Falek A (1988). Neuroimmunomodulation by opiates and other drugs of abuse: relationship to HIV infection and AIDS. Adv. Biochem. Psychopharmacol. 44:145-58. Medline
(23) Donahoe RM, Vlahov D (1998). Opiates as potential cofactors in progression of HIV-1 infections to AIDS. J. Neuroimmunol. 83: 77-87. Medline
(24) Peterson PK, Sharp BM, Gekker G, Portoghese PS, Sannerud K, Balfour HH (1990). Morphine promotes the growth of HIV-1 in human peripheral blood mononuclear cell cocultures. AIDS 4, 869-73. Medline
(25) Carr DJ, Serou M (1995). Exogenous and endogenous opioids as biological response modifiers. Immunopharmacology 31, 59-71. Medline
(26) Hauser KF, Gurwell JA, Martin K, Nath A. (1999). Opiate Drug and HIV Protein Interactions in Striatal Neurons. Neuroimmune Circuits and Infectious disease, 7th Annual Conference-National Institute on Drug Abuse and Society on Disorders of the Neuroimmune Axis, National Institutes of Health, Bethesda, Oct. 7-9.
(27) Hauser KF, Stiene-Martin A, Mattson MP, Elde RP, Ryan SE, Godleske CC (1996). mu-Opioid receptor-induced Ca2+ mobilization and astroglial development: Morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca2+-dependent mechanism. Brain Res. 720: 191-203. Medline
(28) Hauser KF, Harris-White ME, Jackson JA, Opanashuk LA, Carney JM (1998). Opioids disrupt Ca2+ homeostasis and induce carbonyl oxyradical production in mouse astrocytes in vitro: transient increases and adaptation to sustained exposure. Exp.Neurol. 151: 70-76. Medline
(29) Raghavan R, Stephens EB, Koag SV, Adany I, Pinson DM, Li Z, Jai F, Sahni M, Wang C, Leung K, Foresman L, Narayan O (1997). Neuropathogenesis of chimeric simian/human immunodeficiency virus infection in pig-tailed and rhesus macaques. Brain Pathol 7;851-861. Medline
(30) Podell M, Maruyama K, Smith M, Hayes KA, Buck WR, Ruehlmann DS, Mathes LE (1999). Frontal lobe neuronal injury correlates to altered function in FIV-infected cats. J Acquir Immune Defic Syndr 22(1):10-8. Medline
(31) Kolson DL, Lavi E, Gonzalez-Scarano F (1998). The effects of human immunodeficiency virus in the central nervous system. Adv Virus Res 50, 1-47. Medline
(32) Magnuson DS, Morassutti DJ, McBurney MW, Marshall KC (1995). Neurons derived from P19 embryonal carcinoma cells develop responses to excitatory and inhibitory neurotransmitters. Brain Res Dev Brain Res 90(1-2):141-50. Medline
(33) Holden CP, Haughey NJ, Nath A, Geiger JD (1999). Role of Na+/H+ exchangers, excitatory amino acid receptors and voltage-operated Ca2+ channels in human immunodeficiency virus type 1 gp120-mediated increases in intracellular Ca2+ in human neurons and astrocytes. Neuroscience 91(4):1369-78. Medline
(34) Booze RM, Lehner AF, Wallace DR, Welch MA, Mactutus CF (1997). Dose-response cocaine pharmacokinetics and metabolite profile following intravenous administration and arterial sampling in unanesthetized freely moving male rats. Neurotoxicol. Teratol. 19:7-15. Medline
(35) Evans SM, Cone EJ, Henningfield JE (1996) Arterial and venous cocaine plasma concentration in humans: Relationship to route of administration, cardiovascular effects and subjective effects. J. Pharmacol. Exp. Ther. 279:1345-1356. Medline
(36) Hutchings DE, Dow-Edwards D (1991). Animal models of opiate, cocaine, and cannabis use. Clin Perinatol 18: 1-22. Medline
(37) Mactutus CF, Herman AS, Booze RM (1994). Chronic intravenous model for studies of drug (ab)use in the pregnant and/or group-housed rat: An initial study with cocaine. Neurotoxicol. Teratol. 16:183-191. Medline
(38) Wallace DR, Mactutus CF, Booze RM (1996) Repeated intravenous cocaine administration: Locomotor activity and dopamine D2/D3 receptors. Synapse, 23:152-163. Medline
(39) Booze RM, Wood ML, Welch ML, Berry S, Mactutus CF (1999). Estrous cyclicity and behavioral sensitization in female rats following repeated intravenous cocaine administration. Pharmacol. Biochem. Behav. in press.
(40) Darke S, Cohen J, Ross J, Hando J, Hall W (1994). Transitions between routes of administration of regular amphetamine users. Addiction 1994 Sep;89(9):1077-83. Medline
(41) Villemagne V, Yuan J, Wong DF, Danals RF, Hatzidimitriou G, Mathews WB, Ravert HT, Musachio J, McCann UD, Ricaurte GA (1998). Brain dopamine neurotoxicity in baboons treated with doses of methamphetamine comparable to those recreationally abused by human: evidence from [11C]WIN-35,428 positron emmission tomography studies and direct in vitro determinations. J Neurosci 18: 419-427. Medline
(42) Mello NK, Negus SS, Lukas SE, Mendelson JH, Sholar JW, Drieze J (1995). A primate model of polydrug abuse: cocaine and heroin combinations. J Pharmacol Exp Ther 274:1325-37. Medline
(43) Fisher DG, Fenaughty AM, Trubatch B (1998). Seroconversion issues among out-of-treatment injection drug users. J Psychoactive Drugs (3):299-305. Medline
(44) Gerasimov MR, Dewey SL (1999). Gamma-vinyl gamma-aminobutyric acid attenuates the synergistic elevations of nucleus accumbens dopamine produced by a cocaine/heroin (speedball) challenge. Eur J Pharmacol 380(1):1-4. Medline
NeuroAids is a project of Science OnLine
funded through a grant from the National Institute of Mental Health.
|Copyright ©1998 by AAAS Science Publications, Inc.|