Analytic approaches to differential gene expression in AIDS versus control brains.

We previously showed that specific strains of human immunodeficiency virus (HIV)-1 infect the brain and contribute to Neuropathology, Cognitive Distress, and Neuropsychiatric Disease. To study further brain disease that results from HIV-1 infection, we commenced analysis of changes in gene expression in brain. We analyzed RNA purified from Frontal Cortex of 5 HIV-1 infected and 4 HIV-1 negative control subjects RNA was amplified and Affymetrix technology was used to analyze gene expression using the 12,585 gene Affymetrix Human Genome U95A chip. The expressed genes showed highly significant Pearsons correlations with each other within the two groups. Expression intensities were transferred to Microsoft Excel and Spotfire was used to analyze the results. Twenty-group K-means cluster analysis was done for HIV+ and HIV- subjects. Genes that were expressed in the same cluster numbers in the two groups were removed from further analysis. Analysis of Gene expression in the top 13 HIV+ clusters showed expression in the 40 gene categories designated in our prior studies. Genes from several categories occurred in more than one K-means cluster. Genes identified in these lists included several genes that have been previously studied: MBP, Myelin-PLP, NMDA receptor, MAG, astrocytic protein, Notch 3, APP, Senescence, proteasome, Ferritin, signaling, cell cycle, iNOS, Chemokine, splicing, synapse, protein tags, and ribosomal proteins. The first (primary significant) axis of both Principal Component Analyses ordered the genes in the same patient groups as the K-means cluster analysis for the respective patient groups. PCA was thus not more informative than K-Means cluster analysis. Ratios of HIV+ to HIV- intensities were calculated for all the averaged gene expression intensities. The ratio range was 0.14 to 9.26. The genes at the extremes (ad extrema) did not correspond to the gene order by K-means clustering (or PCA). The genes in the top 13 K-means clusters showed low-level changes by expression ratio. Genes ad extrema by ratio were in clusters with very large memberships. Mann-Whitney analysis confirmed expression ratio results. Several inferences result from our preliminary study. First, study design will be different in future studies involving additional replicates. Second, ratios inform us of the extent of changes in gene expression quantitatively. Third, Cluster methodology provides us with more subtle information, how bunches (clusters) of genes behave in terms of their centroids (attractors). Fourth, genes that change extensively by ratio tend to be in the larger k-Means clusters. We conclude that ranking gene expression with the use of expression ratio or by K-means clustering, yield different representations of the data.

Aging and neuro-AIDS conditions and the changing spectrum of HIV-1-associated morbidity and mortality.

Older individuals (>50 years of age) now comprise over 11% of patients with AIDS in the United States. This percentage is expected to continue to grow, due both to the improved longevity of patients prescribed highly active antiretroviral therapy (HAART) and to new infections among older individuals. This review focuses on the neuropsychiatric and neurological conditions that are most likely to be affected by advancing age-HIV-1-associated cognitive-motor disorder, peripheral neuropathy, progressive multifocal leukoencephalopathy, primary CNS lymphoma, and risk for cerebrovascular accident. Age associations with incidence of these disorders and with treatment foci are specified. Implications for future changes in management are discussed.

A bereavement support group intervention affects plasma burden of human immunodeficiency virus type 1. Report of a randomized controlled trial.

OBJECTIVES: This study investigates the potential impact of a bereavement support group on plasma viral load. METHODS: A randomly selected subsample of human immunodeficiency virus type 1 (HIV-1)-positive homosexual men participating in a controlled clinical trial of a bereavement support group intervention was studied. The intervention consisted of one 90-minute group session per week for 10 weeks. The plasma HIV-1 RNA copy number was measured at baseline and after intervention (10 weeks) by the Roche AMPLICOR assay. RESULTS: There was a significant effect of the intervention on the change on the plasma HIV-1 RNA copy number (limited control model, beta = -0.49, p = 0.02; extended control model, beta = -0.37, p = 0.01), independent of antiretroviral therapies; prophylactic therapies against potentially lethal HIV-1 associated conditions; CD4 cell count; viral load; and Centers for Disease Control and Prevention clinical disease stage at baseline. CONCLUSIONS: Bereavement support group interventions may prove to be not only a primary therapy for psychologic distress after bereavement but also an adjunctive therapy for sustained control of plasma viral load in conjunction with highly active antiretroviral therapy in this population.

HIV-1 RNA load in needles/syringes from shooting galleries in Miami: a preliminary laboratory report.

We quantified HIV-1 RNA load in rinses from needles/syringes (N/S) obtained at shooting galleries in Miami and also analyzed the rinses for antibodies for viral proteins. In rinses from 36 N/S that contained visible blood, 14 (39%) had detectable amounts of HIV-1 RNA. Numbers of copies of HIV-1 RNA ranged from the detection limit (400 copies/ml) to 268,000 copies/ml. We also detected antibodies to HIV-1 polypeptides in 34/36 (94%) of rinses from visibly contaminated N/S using Western blots specific for the HIV-1 proteins. No antibodies were detected in laboratory rinses from six visibly clean needles. The presence of HIV-1 RNA in N/S is an important indication of the risk created by N/S sharing as well as by shared paraphernalia and wash waters by injecting drug users.

HIV-1-Associated Cognitive-Motor Disorders: A Research-Based Approach to Diagnosis and Treatment.

The diagnosis of human immunodeficiency virus type 1 (HIV-1)-associated cognitive-motor disorder--either minor cognitive-motor disorder (MCMD) or HIV-1-associated dementia (HAD)--is fraught with potential pitfalls for the clinician. Before making such a diagnosis, clinicians should exclude other etiologies by using neuroimaging, lumbar puncture, and serum chemistries to screen for opportunistic and non-opportunistic infections of the brain and meninges. Clinicians should also consider psychoneurotoxicity (caused from the use of psychoactive substances and prescribed medications) and psychopathology, such as mood, anxiety, and other disorders. In addition, a thorough medical history and physical examination, including a complete neurologic and neuropsychiatric mental status examination, are necessary for an accurate diagnosis. There is also a need for standardized neuropsychological and functional status tests, since the diagnostic criteria for these disorders are partly based on these criteria. Treatment targets should include subclinical cognitive-motor impairment and neuroprotection, as well as MCMD and HAD. Currently, zidovudine remains the best proven treatment for these disorders, but other nucleoside reverse transcriptase inhibitors, as well as nonnucleoside reverse transcriptase inhibitors and protease inhibitors, show promise, and selected agents from these classes are being tested in clinical trials. Other areas that should be investigated are the modulation of inflammatory mediators (such as tumor necrosis factor alpha), neurotransmitter manipulation (especially of dopamine), and nutritional interventions.

Dementia and the Neurovirulence of HIV-1.

Infection with human immunodeficiency virus type 1 (HIV-1) leads rapidly to infection of the brain and subsequent neuropsychological impairment, including subclinical impairment, minor cognitive-motor disorder, and HIV-1-associated dementia (HAD). This article reviews HAD and the factors involved in its pathogenesis; the effectiveness of antiretroviral therapy; the prevalence of HIV-1 and subtypes; and the role of chemokines and cytokines as the capstones associated with neuropathology due to inflammation.

Independent evolution of HIV type 1 in different brain regions.

HIV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V1-V5 domains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, medial temporal lobe, and nonmedial temporal lobe) from three patients. We found that in each separate brain region HIV-1 forms different quasispecies and that there is little gene flow among these regions. In further support of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known amino acid signatures associated with macrophage tropism and the lack of syncytium formation, we found 15 majority amino acid signature patterns from the V1-V5 env sequences associated with the neuroanatomical regions analyzed from the three individuals. Furthermore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be much smaller than genetic distances between brain regions. Specific strains of HIV-1 may be neurotropic or neuroinvasive (replication preference in brain tissue) and may contribute to pathology, cognitive loss, and neuropsychiatric disease.

Oxandrolone, used for treatment of wasting disease in HIV-1-infected patients, does not diminish the antiviral activity of deoxynucleoside analogues in lymphocyte and macrophage cell cultures.

Antiviral agents are the primary therapy for patients infected with HIV-1. However, supportive therapies are often necessary in addition to antiviral drugs because of the devastating wasting process associated with HIV-1 infection and AIDS. Oxandrolone, an anabolic steroid, is used in promoting weight gain and, most important lean body mass (LBM), in patients with HIV-1 disease. We investigated whether oxandrolone interferes with the antiviral activity of zidovudine (ZDV), dideoxyinosine (ddI), and dideoxycytidine (ddC) on HIV-1 replication in peripheral blood lymphocytes and macrophage-monocytes. The nucleoside analogues had nanomolar 50% inhibitory concentrations (IC50) in peripheral lymphocytes. Combinations of nucleoside analogues and oxandrolone did not result in increased IC50 values. Oxandrolone used alone exhibited micromolar IC50 values in peripheral blood lymphocytes. Lack of interference was consistent for nucleoside concentrations up to 5 microM and for oxandrolone concentrations up to 100 microM in several combinations of drugs, viral strains, and peripheral lymphocytes and macrophages. We conclude that oxandrolone can be used for the promotion of weight gain in patients with AIDS-related wasting without interference with the antiviral effects of ZDV, ddI, or ddC.

Intranasal administration of human immunodeficiency virus type-1 (HIV-1) DNA vaccine with interleukin-2 expression plasmid enhances cell-mediated immunity against HIV-1.

DNA vaccine against human immunodeficiency virus type-1 (HIV-1) can induce substantial levels of HIV-1-specific humoral and cell-mediated immunity. To develop more potent HIV-1 DNA vaccine formulations, we used a murine model to explore the immunomodulatory effects of an interleukin-2 (IL-2) expression plasmid on an HIV-1 DNA vaccine following intranasal administration of the combination. When the vaccine and expression plasmid were incorporated into cationic liposomes and administered to mice, the HIV-1-specific delayed-type hypersensitivity response and cytotoxic T lymphocyte activity were significantly increased. Restimulated immune lymphoid cells showed enhanced production of both IL-2 and interferon-gamma and reduced secretion of IL-4. The level of total antibody to HIV-1 antigen was not greatly changed by coadministration of the DNA vaccine and IL-2 expression plasmid. An analysis of serum HIV-1-specific IgG subclasses showed a significant drop in the IgG1/IgG2a ratio in the group that received the plasmid-vaccine combination. These results demonstrate that the IL-2 expression plasmid strongly enhances the HIV-1-specific immune response via activation of T helper type-1 cells.

Morphometric analysis of the peripheral neuropathy of AIDS.

A morphometric study of the peripheral nervous system at autopsy was undertaken in 11 AIDS patients and 10 controls. The left L4, L5, and S1 dorsal root ganglia (DRG) and samples of the sciatic nerve at the buttock, tibial nerve at the knee, and sural nerve at the ankle were collected. Indices of neuronal/axonal degeneration and of segmental demyelination/ remyelination were measured at each level. The small number of cases and evidence of neuropathy in a number of the control cases resulted in statistical significance for only a limited number of comparisons. Nodules of Nageotte in the DRG were increased fivefold in AIDS cases compared with controls, and axonal degeneration in single-teased nerve fibers was increased 9-fold in the sciatic nerve, 28-fold in the tibial nerve, and 12-fold in the sural nerve. The ratios of AIDS to controls for the density of remaining DRG neurons and large myelinated axons were reduced to 0.71 in the DRG, 0.84 in the sciatic nerve, 0.84 in the tibial nerve, and 0.66 in the sural nerve. Axonal regeneration in single-teased nerve fibers was increased threefold at the sciatic nerve level in AIDS, but was markedly reduced at distal levels. Acute segmental demyelination in single-teased nerve fibers was present to a greater extent than in controls at all levels of the peripheral nerves in the AIDS cases. Remyelinating fibers were increased compared with controls only in the proximal sciatic nerve. No case showed the changes of cytomegalovirus infection. In a parallel immunohistochemical study of these AIDS peripheral nerves, T-cell and macrophage infiltration, with cytokine expression, was demonstrated. The pathological process in the neuropathy of terminal AIDS appears to be a multifocal immunologically mediated inflammatory disease, with increased density of macrophages and T cells at all levels of the peripheral nervous system, producing segmental demyelination and axonal degeneration. Reparative processes (axonal regeneration and remyelination) occurred only at the most proximal levels of the nerves.

Medication development of ibogaine as a pharmacotherapy for drug dependence.

The potential for deriving new psychotherapeutic medications from natural sources has led to renewal interest in rain forest plants as a source of lead compounds for the development of antiaddiction medications. Ibogaine is an indole alkaloid found in the roots of Tabernanthe iboga (Apocynaceae family), a rain forest shrub that is native to equatorial Africa. Ibogaine is used by indigenous peoples in low doses to combat fatigue, hunger and in higher doses as a sacrament in religious rituals. Members of American and European addict self-help groups have claimed that ibogaine promotes long-term drug abstinence from addictive substances, including psychostimulants and cocaine. Anecdotal reports attest that a single dose of ibogaine eliminates withdrawal symptoms and reduces drug cravings for extended periods of time. The purported antiaddictive properties of ibogaine require rigorous validation in humans. We have initiated a rising tolerance study using single administration to assess the safety of ibogaine for treatment of cocaine dependency. The primary objectives of the study are to determine safety, pharmacokinetics and dose effects, and to identify relevant parameters of efficacy in cocaine-dependent patients. Pharmacokinetic and pharmacodynamic characteristics of ibogaine in humans are assessed by analyzing the concentration-time data of ibogaine and its desmethyl metabolite (noribogaine) from the Phase I trial, and by conducting in vitro experiments to elucidate the specific disposition processes involved in the metabolism of both parent drug and metabolite. The development of clinical safety studies of ibogaine in humans will help to determine whether there is a rationale for conducting efficacy trials in the future.

Parenteral transmission of HIV among injection drug users: assessing the frequency of multiperson use of needles, syringes, cookers, cotton, and water.

OBJECTIVES: To describe and estimate the frequency of different types of drug preparation and injection practices that could result in the transfer of blood and blood-borne infections among injection drug users (IDUs). METHODS: We analyzed data from interviews administered to 12,323 active IDUs recruited from 19 sites in the United States. The interviews ascertained drug-related behaviors during the previous 30 days. RESULTS: 31.9% of IDUs reported that they engaged in the use of both needle/syringes and cookers/cotton/water previously used by another IDU; 8.6% engaged only in the use of needle/syringes previously used by another IDU; 17.5% engaged only in the use of cookers/cotton/water previously used by another IDU; and 42.0% reported using neither needle/syringes nor cookers/cotton/water previously used by another IDU. Only 12.6% reported use of new (never-used) needle/syringes. The 3935 IDUs who used both needle/syringes and cookers/cotton/water that had been previously used by another IDU had more than 311.000 potential exposures to blood-borne infections from these high-risk practices in 30 days; about 64% of these exposures were from multiperson use of cookers/cotton/water. CONCLUSIONS: Programs to limit parenteral transmission of HIV and other blood-borne infections among IDUs must consider all drug preparation and injection practices that could allow transfer of blood and blood-borne infections among IDUs.

Cocaine enhances monocyte migration across the blood-brain barrier. Cocaine's connection to AIDS dementia and vasculitis?

Cocaine has wide-ranging effects on the immune and neuroendocrine systems (Fiala et al., 1996) resembling an inflammatory "stress" response with upregulation of pro-inflammatory cytokines and stimulation of the HPA axis (Gan et al., 1997). Cocaine abuse has also been associated with vascular pathology, including vasculitis, vasospasm and hemorrhage. These effects suggest that cocaine could perturb the function of endothelial cells, including the blood-brain barrier, and influence the progression to AIDS in HIV-infected individuals (Shapshak et al., 1997; Goodkin et al., 1997). In order to understand clinical consequences of cocaine abuse, it is important to gain insight into molecular and cellular basis of cocaine's effects on immune and endothelial cells. Cocaine's in vitro effects on (a) permeability, (b) immune cell migration, (c) adhesion molecules, and (d) cytokine expression were investigated in a blood-brain barrier model constructed with brain microvascular endothelial cells and fetal astrocytes with the following results: (a) cocaine and tumor necrosis factor-alpha (TNF-alpha) increased the model's permeability to inulin similarly in a dose-responsive fashion; (b) cocaine (10(-4) to 10(-8_ M) enhanced monocyte migration across the barrier with the maximum increase, approximately 100%, by 10(-5) M cocaine; (c) cocaine treatment also increased the expression of endothelial adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecules-1 (VCAM-1) and platelet/endothelial cell adhesion molecule-1 (PECAM-1); (d) although the cocaine in vitro effects on cytokine production by mononuclear cells have been difficult to assess due to a heterogeneity in the degree of responsiveness between individuals, the data suggest that mononuclear cells from cocaine addicts are sensitized to in vitro cocaine challenge with hypersecretion of inflammatory cytokines. Cocaine's in vivo manifestations are compatible with these in vitro effects: (A) chronic cocaine treatment of rats significantly increased rolling white blood cell flux, leukocyte-endothelium adhesion, and ICAM-1 expression in the mesentery (House et al., 1996); (B) cocaine injection to cocaine-dependent subjects tipped the balance of cytokine secretion by mononuclear cells to Th1-type (Gan et al., 1997), and (C) cocaine injection stimulated the hypothalamic-pituitary axis (HPA) to increase both anti- and pro-inflammatory hormonal secretion. Collectively, these results suggest that the immune effects of cocaine on endothelial, immune and neuroendocrine cells impair the function of the blood-brain, barrier, increase cell emigration from the blood vessels, in particular into the brain, and may cause vasculitis. These effects could also increase importation of HIV-1 into the brain.

Cocaine abuse and HIV-1 infection: epidemiology and neuropathogenesis.

The epidemiology of cocaine abuse and potential relationships of cocaine withdrawal to human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are discussed. Neuroendocrinological changes in HIV-1 infection of the central nervous system (CNS) are discussed with the relevant impact of cocaine abuse. HIV-1 load in the brain tissue of infected substance users is described along with possible associations with neuropathology and HAD. Finally, the molecular epidemiology and sequence heterogeneity of HIV-1 and their implications for neuropathogenesis are summarized. The complex context of addressing cocaine abuse in the setting of HIV-1 infection appears more tractable when decomposed into its components.

A bereavement support group intervention is longitudinally associated with salutary effects on the CD4 cell count and number of physician visits.

A randomized, controlled, clinical trial was conducted to examine the impact of a semistructured, 10-week, once weekly, 90-min/session bereavement support group intervention on immunological, neuroendocrine, and clinical health status in human immunodeficiency virus type 1-seropositive (HIV-1+) and HIV-1-seronegative (HIV-1-) homosexual men, compared to a standard of care control condition. A total of 119 homosexual men (74 HIV-1+ and 45 HIV-1-) were assessed at baseline, 10 weeks, and 6 months follow-up. At the 6-month follow-up assessment, the intervention groups exhibited significant beneficial effects compared to controls on changes in CD4 cell, total T-lymphocyte, and total lymphocyte counts, when baseline levels, antiretroviral medication use, CDC stage of disease, and other potentially confounding factors were accounted for. There was no statistically significant effect on the CD4/CD8 ratio or on the CD8 cell count. The effect on CD4 cell count was associated with group attendance and with changes in plasma cortisol level. Plasma cortisol levels decreased significantly among intervention subjects, compared to controls. A significantly reduced number of health care visits over the 6-month follow-up period among the intervention subjects supported the clinical relevance of the immunological changes observed for both HIV-1+ and HIV-1- individuals. These results indicate that behavioral interventions may have salutary immunological and clinical health effects following bereavement among HIV-1-infected individuals. The effect in HIV-1- individuals suggests that this bereavement support group intervention might have similar salutary effects in the general population. Potential effects of such interventions on clinical HIV disease progression are of interest and should be studied.

Heterogeneity of macrophage and T cell subpopulations in peripheral nerves from HIV infected individuals: A preliminary study.

GOAL: To determine the heterogeneity of surface marker expression of macrophages in peripheral nerve of patients who died with AIDS. BACKGROUND: Peripheral neuropathy occurs in 20%-40% of AIDS patients. There is evidence that activated macrophages may be involved in the neural damage associated with HIV-1 infection. We studied the expression of macrophage surface markers CD14, CD11c, CD68, and HLA-DR and also T cell surface markers CD3, CD4, and CD8 in peripheral nerves of AIDS patients. METHODS: Three levels of peripheral nerves (sciatic, tibial, or sural) were examined from a limited number of subjects consisting of 4 HIV-seropositive and 5 HIV-seronegative individuals. Standard immunohistochemical technique utilized alkaline phosphatase conjugate and fuchsin substrate. RESULTS: Surface antigen expression was significantly (p < .0025 increased in HIV-positive tissues compared with HIV-negative controls for CD14 and CD4 in sciatic nerves, CD68 and CD4 in tibial nerves, and CD68 in sural nerves. There were trends for increased expression of HLA-DR, CD3, and CD8 in sciatic nerves, CD11c and CD14 in tibial nerves, and CD14, HLA-DR, and CD4 in sural nerves in HIV-positive tissues compared with HIV-negative controls. CONCLUSION: During the course of AIDS there may be an involvement of all three levels of peripheral nerves suggesting that HIV-related neuropathy is a multifocal process.

HIV-1 proviral DNA load across neuroanatomic regions of individuals with evidence for HIV-1-associated dementia.

A definitive relation between HIV-1 load and the clinical diagnosis of HIV-1-associated dementia (HAD) has not yet been established. Knowledge of the neuroanatomic distribution of HIV-1 load in the brain of individuals with HAD and HIV-1 encephalitis may facilitate elucidation of this relation. Nine individuals with AIDS were analyzed postmortem by three independent methods with each assessment performed blinded to the others: 1) a neuropsychiatric review of clinical records for evidence of possible HAD, 2) HIV-1 DNA load determination by quantitative polymerase chain reaction (PCR) across several neuroanatomic regions, and 3) a pathologic examination for diagnosis of HIV-1 encephalitis by immunohistochemical techniques. Of eight AIDS cases with clinical records sufficient for neuropsychiatric review, seven were shown to have evidence for HAD. HIV-1 DNA was detected and quantified in specimens from all of the medial temporal lobe regions analyzed but was not detectable in the frontal lobe at the same level of sensitivity in two of these cases (<1 per 1000 cellular genomes). HIV-1 DNA load in the medial temporal lobe region was significantly larger than that in the frontal lobe. Only four of seven cases with evidence for HAD were also diagnosed with HIV-1 encephalitis.