Opioid-mediated suppression of interferon-gamma production by cultured peripheral blood mononuclear cells.

Mounting evidence suggests that opiate addiction and stress are associated with impaired cell-mediated immunity. We tested the hypothesis that morphine and the endogenous opioid beta-endorphin (beta-END), a pituitary peptide released in increased concentrations during stress, can suppress the production of the key macrophage-activating lymphokine interferon-gamma (IFN-gamma) by cultured human peripheral blood mononuclear cells (PBMNC). Using a radioimmunoassay to measure IFN-gamma, we found that exposure of PBMNC to biologically relevant concentrations of both opioids significantly inhibited IFN-gamma generation by cells stimulated with concanavalin A and varicella zoster virus. Studies of the mechanism of suppression revealed (a) a classical opioid receptor is involved (suppression was antagonized by naloxone and was specific for the NH2 terminus of beta-END), (b) monocytes are the primary target cell for opioids (monocyte-depleted lymphocyte preparations showed little suppression), and (c) reactive oxygen intermediates (ROI) and prostaglandin E2 are important mediators (scavengers of ROI and indomethacin eliminated the suppression). Based on these findings we suggest that opioid-triggered release of inhibitory monocyte metabolites may play a role in the immunodeficiency associated with narcotic addiction and stress.

Human cytomegalovirus-stimulated peripheral blood mononuclear cells induce HIV-1 replication via a tumor necrosis factor-alpha-mediated mechanism.

Human cytomegalovirus (HCMV) is a potential cofactor in HIV-1 infection. To investigate the mechanism whereby HCMV promotes HIV-1 replication, a PBMC coculture assay which measures HIV-1 p24 antigen release was used as an index of viral replication. HCMV-stimulated PBMC were capable of inducing HIV-1 replication in cocultures with acutely infected PBMC; however, this occurred only when the PBMC were from HCMV-seropositive donors (598 +/- 207 versus 27 +/- 10 pg/ml p24 antigen with PBMC from HCMV-seronegative donors on day 6 of coculture). Upon stimulation with HCMV, PBMC obtained exclusively from HCMV-seropositive donors released tumor necrosis factor (TNF)-alpha (270 +/- 79 pg/ml at 18 h of culture). Monoclonal antibodies to TNF-alpha blocked the activity of HCMV-stimulated PBMC in cocultures both with acutely HIV-1-infected PBMC and with the chronically infected promonocytic line U1. Also, treatment of HCMV-stimulated PBMC with pentoxifylline, an inhibitor of TNF-alpha mRNA, markedly reduced HIV-1 replication in cocultures both with acutely and chronically infected cells. These results indicate that TNF-alpha is a key mediator of HIV-1 replication induced by HCMV-stimulated PBMC and support the concept that this cytokine plays an important role in the pathogenesis of HIV-1 infection.

Functional alterations of swine peripheral blood mononuclear cells by methadone.

In vitro exposure of the synthetic opiate drug methadone allowed evaluation of putative immunomodulatory activities of swine peripheral blood mononuclear cells. Respiratory burst, an index of microbicidal activity, was suppressed by methadone in a dose-dependent manner following exposure for 48 h. The suppression was blocked by the opiate antagonist naloxone. Another macrophage function phagosome-lysosome fusion was impaired by exposure to methadone. A primary lymphocyte-mediated function natural killer cell activity was also affected. In contrast, the macrophage function antibody-mediated phagocytosis was not affected. Because the functions affected by methadone are critical to host defenses against pathogenic organisms, our findings suggest that opiate-mediated immunomodulation merits further study. Moreover, our studies suggest that swine may provide an ideal model for the investigation of opiate-mediated suppression of immune cell functions.

Differential infection of porcine alveolar macrophage subpopulations by nonopsonized Mycobacterium bovis involves CD14 receptors.

The resurgence in mycobacterial infection worldwide has led to renewed attention to the pathogenesis of Mycobacterium species. The purpose of this study was to characterize the infection of alveolar macrophages (AMs) by nonopsonized Mycobacterium bovis, and to elucidate the mechanism by which a differential infection of subpopulations of AM may occur. A difference in susceptibility to Mycobacterium bovis infection of subpopulations of AMs was observed, such that the least dense cells were the least susceptible (21.4 +/- 10.7%) and the most dense cells were the most readily infected (61.8 +/- 5.6%). The percentage of AMs staining for CD14 receptors showed a similar differential distribution, with fewer of the least dense cells expressing CD14 and a greater percentage of the most dense cells staining for CD14 receptor expression. To investigate the role of CD14 receptors in the infection of AMs, anti-CD14 antibody was added to the cell cultures. Infection of AM by Mycobacterium bovis was blocked by up to 60.2% by anti-CD14 antibody but not by isotype control antibody. The results of this study suggest that Mycobacterium bovis selectively infects AM subpopulations, specifically those with the greatest expression of CD14, a putative receptor mechanism for Mycobacterium bovis infection of porcine AM.

Morphine promotes the growth of HIV-1 in human peripheral blood mononuclear cell cocultures.

Because morphine has been shown to alter the function of human T lymphocytes and monocytes, we postulated that morphine would promote the growth of HIV-1 in these cells. To test this hypothesis, a coculture assay was used consisting of phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC) from normal donors and PBMC which had been infected with a viral isolate from an asymptomatic patient, HIV-1AT. The growth of HIV-1AT, as reflected by the concentration of p24 antigen in coculture supernatants, was markedly increased in cocultures that contained morphine. A bell-shaped dose-response curve was observed with three- to fourfold increased growth at a morphine concentration of 10(-12) M. Augmentation of HIV-1AT growth by morphine required an interaction with the PHA-activated donor PBMC. Furthermore, potentiation of HIV-1AT growth by morphine was stereospecific and was antagonized by naloxone and beta-funaltrexamine indicating involvement of an opiate receptor mechanism. These findings provide an additional explanation of how opiates could act as a cofactor in the pathogenesis of HIV-1 in intravenous drug users.

Opioid peptides rapidly stimulate superoxide production by human polymorphonuclear leukocytes and macrophages.

Opioid peptides found in the general circulation can modulate several functions of phagocytic cells that are related to their microbicidal and cytotoxic activity. Since reactive oxygen species are crucial to these activities, the affect of opioid peptides on superoxide (O-2) generation was evaluated with the use of lucigenin-enhanced chemiluminesence (CL). beta-Endorphin and dynorphin stimulate the production of O-2 in human polymorphonuclear leukocytes (PMN) and peritoneal macrophages (PMO) at peptide concentrations that prevail systemically (10(-14)-10(-12)M). There is an inverse dose-response relation for PMN but not PMO. The effect is rapid and sustained in PMN (peak CL at 2-4 min, duration greater than 15 min), whereas it is rapid but brief in PMO (peak 1 min, duration less than 3 min). Naloxone inhibits CL responses by greater than 75% in both cell types.

Benzodiazepines, glia, and HIV-1 neuropathogenesis.

Although the precise mechanisms whereby HIV-1 infection induces neurodegeneration have yet to be determined, a great deal of evidence has incriminated glial cells and the production of proinflammatory mediators in this pathologic process. For this reason, ideal therapeutic agents for the treatment of AIDS dementia would attenuate HIV-1 neuropathogenesis through both direct inhibition of viral expression and suppression of brain cell-produced immune mediators. Benzodiazepines (BDZs), such as Valium, are extensively prescribed drugs for anxiety disorders, which readily cross the blood-brain barrier and have demonstrated immunomodulatory properties. BDZs bind to primary human microglial cells, the principal site of HIV-1 replication in the brain, and inhibit lipopolysaccharide (LPS) induced tumour necrosis factor (TNF-alpha) production by these cells in a concentration-dependent manner. Treatment of HIV-1-infected primary human microglial, as well as mixed glial/neuronal, cell cultures with BDZs inhibits the expression of HIV-1 p24 antigen. BDZ-induced inhibition of HIV-1 expression in chronically infected promonocytic (U1) cells has been found to be associated with decreased activation of the nuclear transcription factor kappa B (NF-kappa B). Because HIV-1 expression is critically dependent on the cellular transcription machinery, inhibition of the activation of transcription factors, which participate in both HIV-1 expression and the production of neurotoxic immune mediators, by BDZ analogs may provide new therapeutic options for AIDS dementia.

U50,488 protection against HIV-1-related neurotoxicity: involvement of quinolinic acid suppression.

The pathogenesis of human immunodeficiency virus type 1 (HIV-1) encephalopathy has been associated with multiple factors including the neurotoxin quinolinate (an endogenous N-methyl-D-aspartate [NMDA] receptor ligand) and viral proteins. The kappa opioid receptor (KOR) agonist U50,488 recently has been shown to inhibit HIV-1 p24 antigen production in acutely infected microglial cell cultures. Using primary human brain cell cultures in the present study, we found that U50,488 also suppressed in a dose-dependent manner the neurotoxicity mediated by supernatants derived from HIV-1-infected microglia. This neuroprotective effect of U50,488 was blocked by the KOR selective antagonist nor-binaltorphimine. The neurotoxic activity of the supernatants from HIV-1-infected microglia was blocked by the NMDA receptor antagonists 2-amino-5-phosphonovalerate and MK-801. HIV-1 infection of microglial cell cultures induced the release of quinolinate, and U50,488 dose-dependently suppressed quinolinate release by infected microglial cell cultures with a corresponding inhibition of HIV-1 p24 antigen levels. These findings suggest that the kappa opioid ligand U50,488 may have therapeutic potential in HIV-1 encephalopathy by attenuating microglial cell production of the neurotoxin quinolinate and viral proteins.

Endomorphin-1 potentiates HIV-1 expression in human brain cell cultures: implication of an atypical mu-opioid receptor.

Endogneous delta and kappa opioid peptides possess a variety of immunomodulatory properties, and kappa-opioid receptor ligands recently were shown to suppress the expression of human immunodeficiency virus type 1 (HIV-1) in microglial cells, the resident macrophages of the brain. To determine whether the newly discovered endogenous mu-opioid receptor ligands endomorphin-1 and -2 would affect HIV-1 replication, these peptides were added to acutely infected brain cell cultures. Endomorphin-1 potentiated viral expression, in a bell-shaped dose-response manner with maximal enhancement approximately equal to 35% at 10(-10) M, in both mixed glial/neuronal cell and purified microglial cell cultures. Endomorphin-1's amplifying effect was blocked by pretreatment of brain cells with either the mu-opioid receptor selective antagonist beta-funaltrexamine or the G protein inhibitor pertussis toxin. However, the classical mu receptor agonists morphine and DAMGO (Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol) had no effect on viral expression or on endomorphin-1's amplifying effect. Taken together, these findings suggest that in this in vitro model of HIV-1 brain infection, endomorphin-1 potentiates viral expression via activation of an atypical mu-selective opioid receptor. They also provide evidence, for the first time, that an endogenous mu-opioid peptide has neuroimmunomodulatory activity.

Arylacetamide-derived fluorescent probes: synthesis, biological evaluation, and direct fluorescent labeling of kappa opioid receptors in mouse microglial cells.

Fluorescein isothiocyanate isomer I (FITC-I) conjugates of 2-(3,4-dichlorophenyl)-N-methyl-N-[1-(3- or 4-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (10 and 14) were prepared either without or with an intervening mono-, di-, or tetraglycyl linker. The 3-substituted fluorescent probes (2-5) were found to retain potent agonist activity in smooth muscle preparations as well as high kappa receptor affinity and selectivity in receptor binding assays. The 4-substituted series (6-9) were substantially less potent than the corresponding 3-substituted compounds. Flow cytometric analysis demonstrated high levels of direct kappa-specific staining of mouse microglial cells by the fluorescent probe 5 containing a tetraglycyl linker, as indicated by a 41% decrease in percent cells positively labeled and a 61% decrease in mean fluorescence intensity in the presence of the kappa-selective antagonist, norbinaltorphimine (norBNI). In similar studies, the probe 2 without a linker exhibited only nonspecific binding. This is the first report of direct, selective staining of kappa opioid receptors by a fluorescent nonpeptide opioid ligand. The results of the present study illustrate the importance of introducing hydrophilic linkers to reduce nonspecific binding of fluorescent probes for opioid receptors.

Cannabinoids and morphine differentially affect HIV-1 expression in CD4(+) lymphocyte and microglial cell cultures.

The influence of substances of abuse on the progression of HIV-1 infection is controversial, and pharmacologic factors have been postulated as a potential explanation for conflicting data arising from epidemiological studies and animal models. In the present study, cell culture models of HIV-1 infection were used to test this hypothesis. The synthetic cannabinoid WIN 55,212-2 was found to potently inhibit HIV-1 expression in a concentration- and time-dependent manner in CD4(+) lymphocyte and microglial cell cultures. In sharp contrast, morphine either inhibited or stimulated viral expression, depending upon the time of drug exposure, and marked differences were observed between CD4(+) and microglial cells. Also, WIN 55,212-2 inhibited the stimulatory effect of morphine in HIV-1 infected CD4(+) cells. These in vitro findings support the notion that pharmacologic factors need to be considered in epidemiological studies and animal models that pertain to HIV-1 infection.

Microglial cell upregulation of HIV-1 expression in the chronically infected promonocytic cell line U1: the role of tumor necrosis factor-alpha.

Culture supernatants from lipopolysaccharide (LPS)-treated murine microglial cells were found to markedly induce the expression of human immunodeficiency virus (HIV)-1 in the chronically infected human promonocytic cell line U1 as detected by measurements of HIV-1 p24 antigen release into U1 culture supernatants. Antibody to tumor necrosis factor (TNF)-alpha had an inhibitory effect on the induction of virus by microglial cell supernatants. Also, treatment of microglia with pentoxifylline, an inhibitor of TNF-alpha production, resulted in suppressed amounts of TNF in the supernatants of LPS-treated microglia and in a reduced stimulatory capacity of these supernatants on HIV-1 expression in U1 cells. These findings support the concept that TNF-alpha production by glial cells plays a pathogenetic role in HIV-1-associated brain disease by promoting the expression of the virus in infected cells.

Morphine amplifies HIV-1 expression in chronically infected promonocytes cocultured with human brain cells.

Previous studies have shown that morphine promotes the replication of human immunodeficiency virus (HIV)-1 in peripheral blood mononuclear cell cocultures. In the present study, we tested the hypothesis that morphine would amplify HIV-1 expression in the chronically infected promonocytic clone U1 when cocultured with lipopolysaccharide-stimulated human fetal brain cells. Marked upregulation of HIV-1 expression was observed in these cocultures (quantified by measurement of HIV-1 p24 antigen levels in supernatants), and treatment of brain cells with morphine resulted in a bell-shaped dose-dependent enhancement of viral expression. The mechanism of morphine's amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor-alpha by microglial cells.

Upregulation of HIV-1 expression in cocultures of chronically infected promonocytes and human brain cells by dynorphin.

Using cocultures of human fetal brain cells and a chronically human immunodeficiency virus-1 (HIV-1)-infected promonocytic line U1, we investigated the effect of dynorphin, an endogenous opioid peptide found in the CNS, on upregulation of HIV-1 expression. Dynorphin and the synthetic kappa receptor agonist U50,488 promoted HIV-1 expression with a bell-shaped concentration-response relationship in which maximal effects were observed at 10(-13) and 10(-11) M, respectively. Pretreatment for 30 min with the kappa receptor antagonist nor-binaltorphimine completely blocked the stimulatory effect of dynorphin and U50,488. The involvement of cytokines on HIV-1 expression was tested. Dynorphin-induced upregulation of HIV-1 in the cocultures was largely blocked by antibodies to tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 but not by antibodies to IL-10. Also, dynorphin stimulated TNF-alpha and IL-6 in the brain cell cultures at both mRNA and protein levels, suggesting the involvement of these cytokines in opioid-induced HIV-1 expression. These findings suggest that endogenous opioid peptides such as dynorphin may have an immunomodulatory function in the CNS and could act as a cofactor in the neuropathogenesis of HIV-1.

Delta-opioid suppression of human immunodeficiency virus-1 expression in T cells (Jurkat).

Delta-opioid receptor (DOR) transcripts and binding sites are expressed by lymphocytes and lymphoid cell lines from several species. Direct modulation of lymphocyte function through DORs affects T cell proliferation, interleukin-2 production, chemotaxis, and intracellular signaling. Moreover, in human DOR-transfected T cells (DOR-Ju.1), delta-opioids have been shown previously to mobilize intracellular calcium rapidly, to inhibit forskolin-stimulated cyclic AMP production, and to activate the mitogen-activated protein kinases ERKs 1 and 2. These observations led us to consider whether delta agonists modify T cell functions, thus affecting the expression of human immunodeficiency virus-1 (HIV-1) by CD4+ T cells. To test this hypothesis, DOR-Ju.1 cells, derived from Jurkat cells stably transfected with a cDNA encoding the neuronal DOR, were stimulated with deltorphin or benzamide, 4-[[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]N- ,[2S[(S*),2alpha,5beta]]-(9Cl) (SNC-80) prior to the addition of HIV-1. Both deltorphin and SNC-80 concentration-dependently inhibited the production of p24 antigen, an index of HIV-1 expression. Inhibition was maximal with 10(-13)-10(-9) M SNC-80 (>60% reduction) or 10(-15)-10(-11) M deltorphin (>50% reduction). At higher concentrations, less inhibition of p24 antigen production was found. Naltrindole (NTI, 10(-11) M), a selective DOR antagonist, abolished the inhibitory effects of 10(-9) M SNC-80, whereas 10(-13) M NTI partially reversed the effect of SNC-80. Thus, activation of DORs expressed by CD4+ T cells significantly (P < 0.05) reduced the expression of HIV-1 by these cells. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.

Kappa-opioid potentiation of tumor necrosis factor-alpha-induced anti-HIV-1 activity in acutely infected human brain cell cultures.

Opioids have been postulated to play an immunomodulatory role in the pathogenesis of HIV-1. Synthetic kappa-opioid receptor (KOR) ligands have been found to inhibit HIV-1 expression in acutely infected microglial cell cultures. We recently found that interleukin(IL)-1beta and tumor necrosis factor(TNF)-alpha have antiviral effects in acutely infected mixed glial/neuronal cell cultures. In the present study, we investigated whether selective KOR ligands would exert antiviral effects in acutely infected brain cell cultures. While the KOR ligand trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl]benze neaceamide methanesulfonate (U50,488) alone had little anti-HIV-1 activity, this opioid potentiated in a concentration-dependent manner the antiviral activity of TNF-alpha, but not of IL-1beta. The potentiating effect of U50,488 was detected after a 6-hr pretreatment and peaked at 24 hr. The KOR antagonist nor-binaltorphimine completely blocked the potentiating effect of U50,488, suggesting the involvement of a KOR-mediated mechanism. Antibodies to TNF-alpha completely blocked the potentiating effect of U50,488, suggesting a critical role for TNF-alpha. Antibodies to IL-1beta blocked the potentiating effect of U50,488, suggesting that IL-1beta was released following U50,488 treatment, which might contribute to the potentiating effect of U50,488. These in vitro findings support the notion that synthetic kappa-opioids could be considered as potential adjunctive therapeutic agents in HIV-1-related brain disease.

Kappa-opioid receptor agonist suppression of HIV-1 expression in CD4+ lymphocytes.

Synthetic kappa-opioid receptor (KOR) agonists have been shown to suppress HIV-1 expression in acutely infected macrophages. In the present study, we examined the effects of the KOR ligand trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl]benzeneaceamide methanesulfonate (U50,488) on HIV-1 expression in CD4+ lymphocytes, the main target cell of this virus. When U50,488 was added to activated CD4+ lymphocytes, HIV-1 expression was inhibited in a concentration- and time-dependent manner with maximal suppression (approximately 60%) at 10(-7) M U50,488. The KOR selective antagonist nor-binaltorphimine (nor-BNI) had no effect by itself on viral expression but blocked the antiviral property of U50,488, suggesting that U50,488 was acting via a KOR-related mechanism. Support for the involvement of KOR was provided by the findings that 34% of activated CD4+ lymphocytes were positive for KOR, using an immunofluorescence technique, and that seven additional synthetic KOR ligands also inhibited HIV-1 expression. The results of this study broaden understanding of the antiviral properties of KOR ligands to include cells outside of the nervous system and suggest a potential role for these agents in the treatment of HIV-1 infection.

Inhibition of swine microglial cell phagocytosis of Cryptococcus neoformans by femtomolar concentrations of morphine.

Microglia are important immune effector cells within the brain. The phagocytosis of nonopsonized Cryptococcus neoformans by swine microglia was used as an in vitro model for studies on cellular mechanisms of opiate-mediated immunomodulation in the brain. Morphine inhibited potently (IC50 approximately 10(-16) M) the phagocytosis of C. neoformans by primary cultures of neonatal pig microglia. The mu opioid agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) also suppressed phagocytosis but with a much lower potency than morphine (IC50 approximately 10(-8) M). The inhibitory effects of morphine and DAMGO were blocked by equimolar concentrations of naloxone and by the selective mu opiate receptor antagonist beta-funaltrexamine. Pertussis toxin but not cholera toxin reversed the inhibitory effects of both morphine and DAMGO. Our data suggest that morphine inhibits phagocytosis of C. neoformans by swine microglia via a mechanism involving mu opiate receptors coupled to a pertussis toxin-sensitive Gi/G(o) protein signaling pathway.

Naltrexone potentiates anti-HIV-1 activity of antiretroviral drugs in CD4+ lymphocyte cultures.

CD4(+) T lymphocytes are the primary cell target for human immunodeficiency virus-1 (HIV-1), and these cells are known to express opioid receptors. Due to the need for new treatment approaches to HIV-1 infection, we sought to determine whether the non-selective opioid receptor antagonist naltrexone would affect HIV-1 expression in CD4(+) lymphocyte cultures and whether naltrexone would alter the antiviral properties of zidovudine (AZT) or indinavir. Activated CD4(+) lymphocytes were infected with a monocytotropic or T-cell tropic HIV-1 isolate, and p24 antigen levels were measured in supernatants of drug-treated or untreated (control) cultures. While naltrexone alone did not affect HIV-1 expression, at a concentration of 10(-12)-10(-10) M naltrexone increased the antiviral activity of AZT and indinavir 2-3-fold. Similar findings with a kappa-opioid receptor (KOR) selective antagonist supported the possible involvement of KOR in naltrexone's potentiation of the antiretroviral drugs. The results of this in vitro study suggest that treatment of alcohol or opiate dependent HIV-1-infected patients with naltrexone is unlikely to interfere with the activity of antiretroviral drugs. Also, based upon naltrexone's safety profile and its synergistic activity in vitro, these findings suggest clinical trials should be considered of naltrexone as an adjunctive therapy of HIV-1 infection.

U50488 inhibits HIV-1 expression in acutely infected monocyte-derived macrophages.

Opioids may play an immunomodulatory role in the pathogenesis of human immunodeficiency virus-1 (HIV-1) infection. Recently, synthetic kappa-opioid receptor (KOR) ligands have been found to have anti-human immunodeficiency virus type 1 activity in acutely infected brain macrophages. In the present study, we investigated whether the selective KOR ligand U50488 would exert such an anti-HIV-1 effect in acutely infected blood monocyte-derived macrophages (MDM). Treatment of acutely infected MDM with U50488 induced a concentration-dependent inhibition of HIV-1 expression. The dose--response relationship of U50488 was U-shaped with a peak effect observed at 10(-13) M, which was evident at both 7 and 14 days post-infection. The KOR antagonist nor-binaltorphimine blocked the anti-HIV-1 effect of U50488 by 73%, indicating involvement of a KOR-mediated mechanism. Also, expression of KOR mRNA and binding activity with a fluorescence-labeled KOR ligand supported the existence of KOR on MDM. Antibodies to the beta-chemokine, RANTES (regulated on activation normal T-cell expressed and secreted), but not to various other cytokines, blocked U50488 inhibition by 56% suggesting that the anti-HIV-1 effect of U50488 involved, in part, the production of RANTES by MDM. Taken together, these in vitro findings support the anti-HIV-1 property of U50488, and suggest that KOR ligands may have therapeutic potential for treating patients with acquired immunodeficiency syndrome.