A new approach to investigating the relationship between productive infection and cytopathicity in vivo.

We describe a novel experimental approach to analyzing virus-host relationships and potential mechanisms of cytopathicity in vivo in simian immunodeficiency virus (SIV) infections. Progressive destruction of lymphoid tissue in the course of infection by SIV or human immunodeficiency virus (HIV) accompanies the loss of CD4+ T lymphocytes and sets the stage for AIDS. Because one of the important early events in this pathological process is lysis of follicular dendritic cells (FDCs), we investigated the controversial role of productive SIV infection in the destruction of FDCs. To differentiate productive infections from the known association of virus with FDCs as immune complexes trapped on cell surfaces, we used detection of spliced viral mRNAs in cells as evidence of productive infection. We found that spliced and unspliced viral RNAs could be detected by in situ hybridization (ISH) with specific antisense oligonucleotide probes in lymphocytes and macrophages with sensitivities of fewer than ten copies of spliced viral RNA per cell. We detected only unspliced RNA in germinal centers where FDCs reside. Thus, no productive infection of these cells can be detected in vivo by this assay, and their destruction likely occurs by indirect mechanisms that have yet to be determined.

Cognitive and motor impairments associated with SIV infection in rhesus monkeys.

Cognitive and motor deficits are now recognized as significant clinical features of infection with the human immunodeficiency virus (HIV). Juvenile rhesus macaques infected with simian immunodeficiency virus (SIV) were found to exhibit cognitive and motor deficits characteristic of HIV infection. Impairment on a motor skill task was the most reliable indicator of infection. Various cognitive impairments were also evident. These deficits were related to SIV infection of the brain but not to inflammatory lesions at a particular locus. The results suggest that the SIV-infected rhesus macaque is a valuable model for understanding the cause of HIV-associated central nervous system dysfunction and for developing a treatment.

Glucocorticoid- and nerve growth factor-induced changes in chromogranin A expression define two different neuronal phenotypes in PC12 cells.

The regulation of chromogranin A mRNA was examined in PC12 cells after treatment with nerve growth factor, dexamethasone, or a combination of the two agents. PC12 cells have low levels of chromogranin A mRNA, and this does not change upon treatment with nerve growth factor. Dexamethasone treatment of these cells results in a 4-fold increase in the amount of chromogranin A mRNA. The dexamethasone-stimulated increase in chromogranin A mRNA is not apparent until at least 16 h after the addition of the drug and is maintained only with continuous culture in the presence of the drug. Dexamethasone and nerve growth factor together increase chromogranin A mRNA to the level seen with dexamethasone alone. Immunohistochemistry shows a similar pattern of protein accumulation within individual cells. Chromogranin B mRNA levels are unaltered by any of the drug treatments described. Treatment with dexamethasone plus NGF seems to be required for full expression of the adrenergic, neuronal phenotype in PC12 cells. Measurement of chromogranin A mRNA provides more specific delineation of neural differentiation and how it is influenced by hormones and growth factors.

The SIV-infected rhesus monkey model for HIV-associated dementia and implications for neurological diseases.

The neuropathogenesis of human immunodeficiency virus (HIV)-associated dementia has remained elusive, despite identification of HIV as the causal agent. Although a number of contributing factors have been identified, the series of events that culminate in motor and cognitive impairments after HIV infection of the central nervous system (CNS) are still not known. Rhesus monkeys infected with simian immunodeficiency virus (SIV) manifest immunosuppression and CNS disease that is pathologically [L. R. Sharer et al. (1991) J. Med. Primatol. 20, 211-217] and behaviorally [E. A. Murray et al. (1992) Science 255, 1246-1249] similar to humans. The SIV model of HIV-associated dementia (HAD) is widely recognized as a highly relevant model in which to investigate neuropathogenesis. With better understanding of neuropathogenesis comes the opportunity to interrupt progression and to design better treatments for HAD. This becomes increasingly important as patients live longer yet still harbor HIV-infected cells in the CNS. The use of the SIV model has allowed the identification of neurochemical markers of neuropathogenesis important not only for HAD, but also for other inflammatory neurological diseases.

Cytopathologic and neurochemical correlates of progression to motor/cognitive impairment in SIV-infected rhesus monkeys.

Neurochemical, pathologic, virologic, and histochemical correlates of simian immunodeficiency virus (SIV)-associated central nervous system (CNS) dysfunction were assessed serially or at necropsy in rhesus monkeys that exhibited motor and cognitive deficits after SIV infection. Some infected monkeys presented with signs of acquired immunodeficiency disease (AIDS) at the time of sacrifice. Seven of eight animals exhibited motor skill impairment which was associated with elevated quinolinic acid in cerebrospinal fluid (CSF). Examination of the brains revealed diffuse increases in glial fibrillary acidic protein immunoreactivity in cerebral cortex in all animals, regardless of evidence of immunodeficiency disease. Reactive astrogliosis preceded or was coincident with the onset of neuropsychological impairments. Virus rescue from CSF of six of eight infected animals showed that one of three animals with AIDS and none of three animals without AIDS at necropsy had virus rescue-positive CSF. Multinucleated giant cells were seen in the brain of only one animal with end-stage AIDS and high systemic virus burden at death. Neither systemic nor CNS virus burden was associated with the onset of CNS dysfunction. SIV-associated motor/cognitive impairment is associated with subtle, widespread changes in CNS cytology and neurochemistry, rather than with large increases in brain virus burden or widespread virus-associated brain lesions.

Synthetic peptides allow discrimination of structural features of CD4(81-92) important for HIV-1 infection versus HIV-1-induced syncytium formation.

Benzylated peptides with a primary amino acid sequence corresponding to either human CD4(81-92) (#18), or chimpanzee CD4(81-92) (#18C), were equipotent inhibitors of human immunodeficiency virus type 1 (HIV-1) infection of CD4+ cells and high-affinity binding of 125I-gp120 to CD4+ cells. The chimpanzee-based CD4(81-92) peptide, however, which differs from the human peptide by a single amino acid substitution (E for G) at position 87, was considerably less potent than the human CD4(81-92)-based peptide congener to inhibit HIV-1-induced cell-cell fusion. These data suggest that a portion of the CD4 molecule contained within the sequence CD4(81-92) is involved in binding gp120 during both HIV-1 infection and HIV-1-induced syncytium formation in human cells, but that the presence of a glutamic acid at position 87 in this sequence is more critical for the CD4/gp120 interaction leading to syncytium formation than for the CD4/gp120 interaction leading to primary infection of CD4-positive cells. The region CD4(81-92) may critically contribute to CD4-mediated HIV-1 pathogenesis in humans, and its alteration might explain the lack of pathogenic sequelae of HIV-1 infection in chimpanzees.

CD4(81-92)-based peptide derivatives. Structural requirements for blockade of HIV infection, blockade of HIV-induced syncytium formation, and virostatic activity in vitro.

CD4(81-92) peptide block human immunodeficiency virus (HIV) infection, virus-induced cell fusion, and antigen production by HIV-1-infected cells when derivatized on specific amino acid residues. An extensive series of structural variants of 1,4,5-tribenzyl-10-acetyl-CD4(81-92) were tested as anti-viral agents in an attempt to define the sequence and derivatization requirements for antiviral activity, and to maximize potency and stability for use as potential therapeutic agents. Alteration of the primary amino acid sequence of the stem compound 1,4,5-tribenzyl-CD4(81-92) diminished or abolished in parallel all three indices of anti-viral activity in a series of altered sequence compounds. Replacement of d- for l-amino acid residues at positions 1, 2, 3, 4, 5, or 6 but not position 10 decreased anti-viral potency, again with parallel effects on infection, synctium formation, and virostatic activity. Omission of the glutamine residue at position 9 did not affect anti-viral potency, while removal of the glutamic acids at position 11 and 12 resulted in virtually complete loss of biological activity. Changes in the derivatization pattern of the CD4(81-92) peptide backbone also affected anti-viral potency and efficacy. Optimal activity was obtained with benzyl residues at positions 1, 4, and 5, whereas the 1,4,7-tribenzyl-CD4(81-92) compound was without activity in all assays tested. Replacement of one of the benzyl groups with an acetamidomethyl moiety resulted in complete loss of biological activity. The previously reported (Nara et al., Proc Natl Acad Sci USA 86: 7139-7143, 1989) virostatic activity of 1,4,5-tribenzyl-10-acetyl-CD4(81-92) (peptide #18) is apparently due to acetylation, since the desacetyl stem compound shows much less virostatic activity while still possessing full anti-infective and anti-syncytial activity, and acetylation of the N-terminus rather than the lysine of 1,4,5-tribenzyl-CD4(81-92) yields a virostatic compound equipotent to peptide #18. Cyclization of the tribenzyl peptide to further conformationally restrict the molecule resulted in a compound with anti-infection, anti-syncytial, and virostatic activity at submicromolar concentrations.

Peptides derived from the CDR3-homologous domain of the CD4 molecule are specific inhibitors of HIV-1 and SIV infection, virus-induced cell fusion, and postinfection viral transmission in vitro. Implications for the design of small peptide anti-HIV therapeutic agents.

Peptides 12-25 amino acids in length from the V1J1 region of the CD4 molecule (residues 1-120) were synthesized as randomly derivatized, deliberately derivatized, or pure peptide products, and tested for their ability to inhibit HIV-1-induced cell fusion, HIV-1 and SIV infection of CD4-positive human cells, HIV-1 envelope glycoprotein binding to the CD4 molecule, CD4-neutralizing antibody binding to the CD4 holoreceptor, and CD4-dependent cellular immune function in the mixed lymphocyte and cytotoxic T-cell bioassays. Only peptides derived from the complementarity-determining region 3 (CDR3)-homologous domain of CD4, in particular CD4(81-92) and CD4(81-101), were effective antiviral agents. Within the CD4(81-92) series, R-group derivatization of selective amino acid residues was an absolute requirement for biological activity. The prototype compound T1C4E5-tribenzyl-K10-acetyl-TYICEVEDQKEE inhibited HIV-1-induced cell fusion at 32 microM, HIV-1 infection of CEM-SS cells at 10 microM, SIV infection of CEM-174 cells at less than 125 microM, gp120/CD4 binding at 60 microM, and postinfection cell-mediated viral transmission at 10-15 microM. Compounds of identical structure and derivatization, but of altered primary sequence, were substantially less active, or without activity, in these assays. These data indicate that the effect of amino acid derivatization of the CD4(81-92) peptide was most likely restriction of the flexible underivatized peptide backbone to a conformation closely approximating that of the CDR3-homologous gp120 binding site of the native CD4 molecule. Peptide antiviral activity was specific, as judged by lack of cytotoxicity, lack of inhibition of HTLV-1-induced cell fusion, and lack of inhibition of CD4-dependent cellular immune function in vitro. Further derivatization of the prototype compound involving the production of cyclic congeners yielded peptides with submicromolar potency to block HIV-1 infection, strengthening the hypothesis that previous peptide derivations accomplished partial restriction of the conformation of CD4(81-92) to one favorable for interaction with gp120. Concentrations of the original prototype compound T1C4E5-tribenzyl-CD4(81-92) that inhibited infection in vitro more than 50% could be achieved for several hours by intravenous infusion in primates and were well-tolerated at these levels. The peptide was not efficacious to inhibit establishment of viral infection at these doses; however, peptide treatment did lower average viral antigenemia and delay the cumulative time to morbidity relative to the control group.

Neuroscience research in AIDS.

1. The human immunodeficiency virus invades the central nervous system early after infection where it later gives rise to cognitive, motor, and behavioral manifestations in children and adults. 2. Ranging from mild impairments to frank dementia, CNS manifestations can be diagnosed and measured with standard neuropsychological test batteries. 3. Great strides have been made with treatment: CNS manifestations are treatable, as are depression, psychosis, and delirium which sometimes accompany HIV disease at different stages. 4. With startling advances in antiretroviral therapy and lower mortality, patients face a constellation of new concerns stemming from HIV's transformation to a more chronic disease. 5. There are many compelling research directions ahead, including the psychosocial impact of living with HIV as a chronic disease, the development of medications expressly targeted to the CNS, and basic research on neuropathogenesis, including trafficking of virus into the CNS.

In vivo test system for tumor production by cell lines derived from lower vertebrates.

The immune suppressed lizard, Anolis carolinensis, can be used to test for in vivo tumor production by cell lines derived from a variety of ectothermic vertebrates. Cell lines tested for tumor production were also assessed for loss of attachment-dependent proliferation and contact inhibition of cell overlap. The results demonstrate that the criteria standardly used to assess transformation and neoplastic change in cultured mammalian cells apply equally well to cultured cells from ectotherms.

HIV in the CNS: pathogenic relationships to systemic HIV disease and other CNS diseases.

Research on the pathogenesis of the human immunodeficiency virus (HIV) infection of the central nervous system (CNS) has reached a pivotal stage. While the incidence of HIV dementia appears to be declining, the prevalence of milder, yet debilitating, neuropsychological impairments may rise as individuals infected with HIV live longer. There are also concerns about CNS reservoirs of latently infected cells. Building upon progress in understanding HIV neuropathogenesis, the time is ideal to expand research on the interrelationships between the CNS and systemic HIV disease, and extend the boundaries of this research to the neuropathogenic similarities between HIV and other CNS inflammatory diseases. Neuropathogenic insights gained from these pursuits can spawn new treatment strategies for HIV/CNS disease as well as potentially other diseases of the nervous system.

An early increase in somatostatin mRNA expression in the frontal cortex of rhesus monkeys infected with simian immunodeficiency virus.

Motor and cognitive impairment is common in human immunodeficiency virus disease in humans and simian immunodeficiency virus (SIV) disease in rhesus monkeys. We have examined peptide neurotransmitter expression in the frontal cortex of SIV-infected rhesus monkeys to identify alterations in cortical neurons that might explain this impairment. A 2-fold higher number of preprosomatostatin (SRIF) mRNA-positive interneurons was observed in layer IV of frontal cortex in two separate cohorts of SIV-infected animals compared to uninfected controls. Increased SRIF mRNA expression in layer IV was independent of clinical signs of immunodeficiency disease and was associated with both motor and cognitive impairment. Altered SRIF mRNA expression in deeper cortical layers was associated specifically with motor impairment. Increased SRIF mRNA expression occurred without detectable changes in cortical cell density. These data suggest two mechanisms for cortical dysfunction associated with lentivirus infection. Increased SRIF mRNA expression in layer IV may be due to altered patterns of activity in cortical afferents that project to layer IV, while increased SRIF mRNA expression in deeper cortical layers could reflect susceptibility to locally generated mediators in response to primate lentivirus infection of the brain. Altered function of somatostatinergic interneurons may contribute to primate lentivirus-induced encephalopathy.

Effects of chronic zidovudine administration on CNS function and virus burden after perinatal SIV infection in rhesus monkeys.

Continuous intravenous administration of zidovudine (AZT) has been reported to improve cognitive function in HIV-infected pediatric patients (Pizzo et al., 1988). The effects of long-term zidovudine treatment in the perinatally infected pediatric population, including antiviral efficacy and effects on cognitive and motor function has not been systematically examined. These questions were addressed in rhesus macaque infants infected at birth with SIVSMM/B670, a primate model for infantile HIV infection and disease (Eiden et al., 1993a). Continuous or intermittent administration of AZT during the first 6 months following infection resulted in about a doubling of lifespan, a delay in the occurrence of motor impairment, and lower virus burden and quinolinic acid levels in cerebrospinal fluid (CSF) following administration of the antiviral drug.

Neuronal substrates for SIV encephalopathy.

Prior to the onset of immunodeficiency disease, neurochemical and neuropathological events associated with motor and/or cognitive impairment can be identified in rhesus monkeys infected with simian immunodeficiency virus (SIV). These are astrocytosis, up-regulation of mRNA encoding the neuropeptide somatostatin (SRIF) and an increased expression of MHC Class II antigen. End-stage immunodeficiency disease has been associated with robust viral expression in the CNS frequently observed as multinucleated giant cell formation. SIV encephalitis has not been observed in animals whose only clinical signs of SIV disease were motor and/or cognitive impairment. These data suggest that neuronal dysfunction discernable as altered neuropeptide expression in cortical neurons precedes frank structural damage to the CNS in SIV encephalopathy. This model is consistent with the mechanism of neuropathogenesis in human HIV encephalopathy that can be partially inferred from neurochemical and neuropathological examination of autopsy material in HIV disease.

Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras, or src tyrosine kinase.

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.

Enhanced expression of Ca2+ channels by nerve growth factor and the v-src oncogene in rat phaeochromocytoma cells.

1. Rat phaeochromocytoma (PC12) cells were used to investigate the expression of Ca2+ channel types during neuronal differentiation. Neuronal differentiation was induced by treatment with nerve growth factor (NGF) or by activation of a temperature-sensitive tyrosine kinase (pp60v-src) in genetically modified PC12 (PC12/v-src) cells. PC12 cells differentiated morphologically in the presence of NGF. When grown at the permissive temperature of 37 degrees C which activates the kinase activity of pp60v-src, PC12/v-src cells differentiated morphologically with the extension of neurites. In contrast, PC12/v-src cells grown at the non-permissive temperature of 40 degrees C continued to divide and were morphologically indistinguishable from control PC12 cells. 2. Whole-cell Ca2+ currents were measured in PC12 cells using Ba2+ as the charge carrier. Ba2+ currents measured at the peak of the current-voltage curve from a holding potential of -80 mV were -0.28 +/- 0.04 nA (mean +/- S.E.M.) in control PC12 cells compared to -1.25 +/- 0.16 nA in NGF-differentiated cells. The current density increased from 9.4 +/- 0.7 pA/pF in control PC12 cells to 22.8 +/- 2.4 pA/pF in NGF-differentiated PC12 cells. Ba2+ currents were -0.24 +/- 0.04 nA in undifferentiated PC12/v-src cells grown at the non-permissive temperature of 40 degrees C compared to -0.95 +/- 0.16 nA in differentiated PC12/v-src cells grown at the permissive temperature of 37 degrees C. The current density increased from 4.5 +/- 0.5 pA/pF in PC12/v-src cells grown at the non-permissive temperature of 40 degrees C to 13.3 +/- 2.4 pA/pF in PC12/v-src cells grown at the permissive temperature of 37 degrees C. 3. The sensitivity of Ba2+ currents to omega-conotoxin GVIA (omega-CgTX) was determined for currents measured at the peak of the current-voltage curve (0 mV in 10 mM Ba2+) from a holding potential of -80 mV. In NGF-differentiated PC12 cells, 10 microM omega-CgTx inhibited 68.1 +/- 3.2% of the total Ba2+ current compared to 35.9 +/- 4.1% in control cells. The density of the omega-CgTX-sensitive current increased from 3.3 +/- 0.4 pA/pF in control cells to 15.7 +/- 2.0 pA/pF in NGF-differentiated cells. In differentiated PC12/v-src cells grown at 37 degrees C, omega-CgTX inhibited 52.2 +/- 4.2% of total Ba2+ current compared to 41.1 +/- 3.8% in PC12/v-src cells grown at 40 degrees C. The density of the omega-CgTX-sensitive current increased from 1.9 +/- 0.3 to 7.4 +/- 2.0 pA/pF with v-src-mediated differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Zidovudine treatment prolongs survival and decreases virus load in the central nervous system of rhesus macaques infected perinatally with simian immunodeficiency virus.

To assess the potential therapeutic effects of zidovudine, rhesus macaques were inoculated with simian immunodeficiency virus (SIV) strain SMM/B670 at birth and infused either continuously or intermittently with zidovudine for 6-7 months. Zidovudine did not prevent infection but did significantly increase survival time, which was associated with lower serum p26 viral core antigen levels, a lower virus burden in the cerebrospinal fluid (CSF), and lower CSF quinolinic acid levels than in untreated monkeys. Two of 5 infected, untreated monkeys developed motor impairment within 6 months following infection, whereas motor impairments did not occur in infected, zidovudine-treated monkeys until after the drug was discontinued. Zidovudine treatment was well tolerated by rhesus infants with minimal, transient side effects. These results demonstrate that zidovudine treatment significantly decreases virus load within the central nervous system (CNS) and delays the onset of CNS dysfunction and immune disease in rhesus monkeys perinatally infected with SIV.

Simian immunodeficiency virus burden in tissues and cellular compartments during clinical latency and AIDS.

In the course of human immunodeficiency virus infection or of the related simian immunodeficiency virus (SIV), progression to AIDS is associated with high virus burdens in blood. How virus burden in the bloodstream is related to virus burden in tissue reservoirs was addressed in an animal model of rhesus macaques infected with SIV. In situ hybridization and quantitative image analysis were used to quantitate virus burden. Animals who developed AIDS had high levels of virus production and storage in lymphoid tissue reservoirs and evidence of productive infection of macrophages in the nervous system. With the quantitative approach described, it should be possible to design and assess the impact of treatment and shed light on the outstanding issues in pathogenesis.

Perspectives from the National Institute of Mental Health: Preventing or living with aids.

This article provides a succinct overview of the history and current and future research priorities of the Office on AIDS at the National Institute of Mental Health (NIMH). Throughout its history and currently, the Office on AIDS has encouraged and supported research on primary prevention of human immunodeficiency virus (HIV) transmission, effects of HIV disease on the central nervous system, and coping with the sequelae of infection. Future directions for the NIMH include the dissemination of research fmdings to the community, investigation of mechanisms for involving and retaining participants in large-scale vaccine trials, and continued attention to the prevention of HIV transmission through behavior change.