Newborn Hearing Screenings in Human Immunodeficiency Virus-Exposed Uninfected Infants.

Perinatal HIV infection and congenital cytomegalovirus (CMV) infection may increase the risk for hearing loss. We examined 1,435 infants enrolled in the Surveillance Monitoring of ART Toxicities (SMARTT) study of the Pediatric HIV/AIDS Cohort Study (PHACS) network, a prospective study of the safety of in utero antiretroviral (ARV) exposures. We determined the proportion of perinatally HIV-exposed uninfected (HEU) newborns who were referred for additional hearing testing, and evaluated the association between in utero ARV exposures and newborn hearing screening results. Using a nested case-control design, we also examined congenital CMV infection in infants with and without screening referral. Congenital CMV infection was determined based on CMV DNA detection using a nested PCR assay in peripheral blood mononuclear cells obtained within 14 days of birth. Among the 1,435 infants (70% black, 31% Hispanic, 51% male), 45 (3.1%) did not pass the hearing screen and were referred for further hearing testing. Based on exact logistic regression models controlling for maternal use of tobacco and ototoxic medications, first trimester exposure to Tenofovir was associated with lower odds of a newborn hearing screening referral [adjusted odds ratio (aOR) = 0.41, 95% confidence interval (CI): 0.14-1.00]. Exposure to Atazanavir was linked to higher odds of newborn screening referral, although not attaining significance [aOR = 1.84, 95% CI: 0.92-3.56]. Maternal ARV use may have varying effects on newborn hearing screenings. These results highlight the importance for audiologists to be knowledgeable of in utero ARV exposures in HEU children because of the possibility of higher referrals in these children.

Cotransduction of nondividing cells using lentiviral vectors.

Diseases such as AIDS and cancers may require the introduction of multiple genes into either stem cells or nondividing cells, among others, for therapeutic purposes. Such genes may act at different points of the disease pathway, or may constitute a regulatory loop to bypass or rectify the defective gene or pathway underpinning the disease. Ideally, the therapeutic genes must be transduced together in diverse combinations, and the introduction should occur without constraints. Since lentiviral vectors can transduce both dividing and nondividing cells, they are ideal vehicles to investigate combinatorial gene transfer into diverse cells. In this study, we demonstrate that by using two independent lentiviral vectors, pseudotyped with the protein g of vesicular stomatitis virus, up to four genes can be introduced simultaneously into single dividing and nondividing cells. Up to 45% and 73% of dividing and nondividing cells, respectively, could be transduced with two lentiviral vectors. The efficiency of cotransducing a single cell was the product of the individual transduction efficiencies and suggested the absence of viral interference. Multiple and combinatorial gene transduction using lentiviral vectors may prove useful in gene therapy.

Lymphoproliferative responses to recombinant HIV-1 envelope antigens in neonates and infants receiving gp120 vaccines. AIDS Clinical Trial Group 230 Collaborators.

Children of mothers infected with human immunodeficiency virus type 1 (HIV-1) were immunized at birth and at 1, 3, and 5 months with 1 of 3 doses of recombinant gp120 vaccines prepared from SF-2 or MN strains of HIV-1. A total of 126 children were not infected; 21 received adjuvant only. Vaccine recipients developed lymphoproliferative responses on >/=2 occasions, responding more often to homologous HIV-1 antigens than did adjuvant recipients (56% vs. 14%; P<.001). Responses were appreciated after 2 immunizations and were maintained for >84 weeks after the last immunization. An accelerated immunization schedule (birth, 2 weeks, 2 months, and 5 months) with the lowest dose of the SF-2 vaccine produced responses in all 11 vaccinees by 4 weeks. Responses to heterologous envelope antigens were also detected. Immune responses to vaccination are achievable at an age when some infection (perinatal or breast milk exposure related) may be prevented.

Cerebrospinal fluid HIV RNA originates from both local CNS and systemic sources.

OBJECTIVE: To identify the sources of HIV virions in CSF by modeling treatment-associated HIV dynamics. BACKGROUND: We postulated a model in which cell-free CSF virions originate from two major sources, namely, systemic non-CNS and CNS tissues, the latter including brain parenchyma and meninges. The model predicted that with initiation of antiretroviral therapy, the acute-phase decline in CSF HIV RNA levels would be controlled by the kinetics of the dominant virion source (systemic versus CNS). Based on prior observations, we hypothesized that the dominant source of CSF virions would shift from systemic to CNS in more advanced disease. METHODS: Three patient groups were studied: Group 1 (n = 5): nondemented, with early HIV disease (CD4+ lymphocytes > or = 400/microL) or pleocytosis (CSF leukocytes > or = 4/microL); Group 2 (n = 5): nondemented, with advanced HIV disease (CD4+ < 400/microL) and no pleocytosis; Group 3 (n = 2): patients with HIV-associated dementia (HAD). All patients began a new, highly active antiretroviral treatment regimen and underwent serial lumbar punctures and phlebotomies. RESULTS: For patients in Group 2, the rate of decline in CSF HIV RNA was slower than in plasma (p < 0.00001). For Group 1, the rate of decline in CSF was not different from plasma (p > 0.25). Patients with HAD showed high CSF HIV RNA after 5 to 6 weeks of treatment despite a 100-fold decrease in plasma HIV RNA. CONCLUSIONS: CSF and plasma HIV dynamics became increasingly independent in advanced HIV disease, and the compartmental discrepancy was largest in HAD. Our findings suggest that viral replication in CNS tissues may constitute a major, independent source of CSF HIV RNA. In patients with HAD, brain parenchyma itself may be the principal CNS tissue source, and CNS-targeted treatment strategies may be required to eradicate this infection.

Lack of autologous neutralizing antibody to human immunodeficiency virus type 1 (HIV-1) and macrophage tropism are associated with mother-to-infant transmission.

To investigate factors that affect mother-to-infant transmission of human immunodeficiency virus type 1 (HIV-1), autologous neutralizing antibody, viral load, and viral tropism were evaluated in 28 pregnant women infected with HIV-1, of whom 8 were transmitters and 20 nontransmitters. One (12%) of 8 transmitters versus 11 (55%) of 20 nontransmitters had autologous neutralizing antibody (P=.04). Plasma levels of HIV-1 RNA and infectious HIV-1 titers (mean+/-SD) in peripheral blood mononuclear cells (PBMC) at delivery did not differ significantly between transmitters and nontransmitters (24, 266+/-10,101 vs. 31,589+/-9128 copies/mL and 29+/-12 vs. 42+/-17 infected cells per 106 PBMC, respectively). However, only transmitters (4 [50%] of 8) were HIV p24 antigen positive. The ability of HIV-1 strains to induce syncytium did not differ between groups (P=.6); however, only non-syncytium-inducing isolates were transmitted. Isolates from 4 (80%) of 5 transmitters versus 2 (18%) of 12 nontransmitters (P=.03) demonstrated increasing replication in macrophages. Thus, lack of autologous neutralizing antibody and increased replication in macrophages were significantly associated with mother-to-infant transmission. In addition, autologous neutralizing antibody was associated with reduced viral load.

Human insulin receptor and insulin signaling proteins in hepatic disease.

Insulin regulates hepatocellular metabolism and growth following insulin receptor (IR) autophosphorylation and activation of the intracellular adapter protein, insulin receptor substrate 1 (IRS-1). IRS-1 activates SH2 domain proteins such as Grb2, which may be vital to hepatocyte growth. To determine if these substances are abnormally expressed under pathophysiologic conditions, IR, IRS-1, Grb2 protein, and IR mRNA were studied in normal human liver (n = 10), cirrhotic liver (n = 10), and hepatocellular carcinoma (HCC) (n = 10) that had been procured during operative procedures. IR mRNA was quantified by S1-nuclease assay using a 195-bp digoxigenin-labeled IR DNA probe and normalized to the level of expression of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Protein concentrations were determined by immunoblot analysis following SDS-PAGE of liver homogenate samples. Labeled DNA and antibody-complexed protein were detected by chemiluminescent means and quantified by densitometric analysis (mean densitometric units +/- standard error). Similar levels of IR mRNA were observed in normal tissue, cirrhosis, and HCC. IR protein concentration was significantly greater in HCC than in normal liver (1.82 +/- 0.2 vs 1. 25 +/- 0.17; P < 0.05). IRS-1 was significantly increased in cirrhosis compared to normal liver (1.61 +/- 0.31 vs 0.86 +/- 0.21; P < 0.05). No differences were observed in Grb2 in the three tissue types. Insulin receptor overexpression, previously seen in other tumor types, may confer an insulin-mediated growth advantage in HCC if added receptors reflect functional high affinity binding sites. Although an altered mass of IRS-1 protein was not observed in HCC, an IRS-1 increase in cirrhosis may favor hepatic regeneration.

Overexpression of hepatic pancreatic polypeptide receptors in chronic pancreatitis.

Pancreatic polypeptide (PP) receptors have recently been demonstrated on liver microsomal membranes although the mechanisms of PP action on hepatocytes remain uncertain. The binding characteristics of these high affinity receptors under pathophysiologic conditions were studied in rats with oleic acid-induced chronic pancreatitis (CP), a state associated with diminished pancreatic PP content. Sixteen pancreatitic and 11 sham-operated control animals either were 16-h fasted or were given free access to food prior to organ removal. Competitive binding studies were performed by incubating hepatocyte microsomal preparation with 125I-labeled PP (20-40 pM) and increasing concentrations of nonlabeled PP (1 x 10(-10) to 1 x 10(-6) M). After total and nonspecific binding was quantified by gamma counting, coefficients of dissociation (Kd) and maximal binding sites (Bmax) were determined by Scatchard analysis of specifically bound radioactivity. Binding data were normalized to membrane protein content and expressed as means +/- standard error. Bmax was significantly greater in tissue from fed control animals than from fasted controls (4.46 +/- 0.36 versus 2.83 +/- 0.25, P < 0.05). Bmax was significantly greater under fasted conditions in tissue from CP animals than from controls (5.25 +/- 0.94 versus 2.83 +/- 0.25, P < 0.01). Under fed conditions, this differences was abolished by the increase in maximal binding in the control group. The fasting-associated decrease in maximal binding sites observed in controls did not occur in CP specimens. Increased Bmax in fed versus fasted control, as well as fasted CP versus fasted control, were associated with slight reciprocal decreases in receptor affinity. These data indicate that hepatic PP receptor concentration is upregulated in this model of chronic pancreatitis, most likely due to diminished exposure to ligand. Furthermore, normal PP receptor responses to the fed/fasted state are blunted in this condition. Regulatable PP receptor changes may play a role in altered hepatic metabolism previously observed in chronic pancreatitis.

Gene therapy targeting cord blood-derived CD34+ cells from HIV-exposed infants: preclinical studies.

Hematopoietic CD34+ cells from placental and umbilical cord blood (PUCB) can be valuable vehicles for gene therapy of immunodeficiencies and genetic disorders. We have conducted preclinical studies towards the treatment of HIV-1-infected infants with genetically 'immunized' CD34+ cells derived from PUCB using anti-HIV-1 hairpin ribozyme genes. PUCB was collected from 10 newborns of HIV-1-positive mothers. CD34+ cells were enriched with a modified procedure using Dynal immunomagnetic beads and chymopapain, stimulated with stem cell factor (SCF), IL-3 and IL-6, and transduced using cell-free recombinant retroviral vector (MJT) expressing a ribozyme against the U5 region of HIV-1. No significant differences were observed in the growth pattern of CD34+ cells from normal infants, HIV-1 exposed infants or infants confirmed to be infected by HIV-1. The transduction efficiency of the CD34+ cells from all the infants was also comparable. MJT-transduced CD34+ cells from an HIV-1-infected infant were maintained in a liquid culture system for 4 weeks, and the progeny macrophage cells were challenged with the monocyte-tropic laboratory strain, HIV-Bal, or the HIV-1 isolate from the infant's mother. Significant inhibition of virus replication was observed in ribozyme-transduced cells. Thus, we have demonstrated efficient and stable gene transfer into progenitor cells from the cord blood of HIV-1-exposed or -infected infants and shown that protection from HIV-1 infection was conferred to the progeny cells produced by CD34+ cells transduced with the anti-HIV ribozyme gene construct.

Predictive value of quantitative plasma HIV RNA and CD4+ lymphocyte count in HIV-infected infants and children.

CONTEXT: Pediatric human immunodeficiency virus (HIV) infection has unique viral pathogenetic features that preclude routine extrapolation from adult studies and require specific analysis. OBJECTIVES: To evaluate the prognostic value of 2 key laboratory markers-plasma RNA and CD4+ lymphocyte count-for HIV disease progression in infants and children and to establish targeted values for optimal outcome. DESIGN: Data from a cohort of 566 infants and children who participated in a randomized, placebo-controlled trial of nucleoside reverse transcriptase inhibitors (ACTG 152) were analyzed. The trial was conducted between 1991 and 1995 and enrolled a heterogeneous cohort of antiretroviral therapy-naive children (age, 3 months to 18 years); patients had a median follow-up of 32 months. MAIN OUTCOME MEASURES: The trial clinical end points consisted of time to first HIV disease progression (growth failure, decline in neurologic or neurodevelopmental function, opportunistic infections) or death. RESULTS: Baseline plasma RNA levels were high (age group medians, 5 x 10(4) to >10(6) copies/mL), and both baseline RNA and CD4+ lymphocyte count were independently predictive of subsequent clinical course. Risk reduction for disease progression between 49% and 64% was observed for each log10 reduction in baseline RNA and was linear without suggestion of a threshold or age effect. Disease progression predictive power was enhanced by the combined use of plasma RNA and CD4+ cell count. Marker values of less than 10000 copies/mL for plasma RNA and greater than 500 x 10(6)/L (<6.5 years of age) or greater than 200 x 10(6)/L (>6.5 years) for CD4+ cell count were associated with a 2-year disease progression rate of less than 5%. CONCLUSIONS: Two key laboratory markers--plasma RNA and CD4+ lymphocyte count-are independent predictors of clinical course among HIV-infected infants and children. The linear, age-independent relationship between log10 plasma RNA and relative risk of disease progression strongly supports therapeutic efforts to achieve plasma virus levels as low as possible.

HIV-induced apoptosis of activated primary CD4+ T lymphocytes is not mediated by Fas-Fas ligand.

OBJECTIVE: To investigate the role of the Fas-Fas ligand (FasL) interaction in HIV-1-induced apoptosis of primary CD4+ T lymphocytes. DESIGN: Activated CD4+ T lymphocytes are the main target of HIV, and T-cell activation leads to the expression of Fas-FasL and enhances HIV-mediated apoptosis. Phytohemagglutinin-activated primary CD4+ T cells were infected with HIV; the process of cell death was examined, and whether the dying and dead cells were the productively infected cells. The modulation of Fas and FasL expression and its role in HIV-induced cell death was also investigated. METHODS: The number of viable and dead cells was determined by trypan blue exclusion. Apoptosis was quantified using an enzyme-linked immunosorbent assay measuring the release of cytoplasmic histone-associated DNA fragments. The percentage of HIV-infected cells was determined by FACS analysis, and viral production was assessed by a p24 core antigen assay. The following three markers, HIV-gp-120, annexin-V and 7-AAD, were used to monitor the apoptotic process in HIV-negative and positive cells. Fas and FasL expression was analyzed at the RNA level by reverse transcription polymerase chain reaction and at the protein level by flow cytometry. The contribution of Fas-FasL interactions to apoptosis was examined by blocking experiments using the antagonist ZB4 anti-Fas antibody. RESULTS: HIV-induced apoptosis in activated purified CD4+ T lymphocytes required infectious virus and was dose-dependent. Apoptosis in HIV-infected cultures was mostly confined to productively infected cells. The expression of Fas and FasL was not significantly modulated by infection and blocking Fas-FasL interactions did not reduce the extent of apoptosis. CONCLUSIONS: HIV-induced apoptosis of activated CD4+ T cells in vitro is confined to productively infected cells and is not mediated by a Fas-FasL interaction.

Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response. ACTG 241 Protocol Virology Substudy Team.

BACKGROUND: CD4+ lymphocyte counts and plasma HIV-1 RNA levels predict progression of HIV-related disease, but the relative importance of these and other virological factors in defining response to antiretroviral therapy is not yet clear. OBJECTIVE: To determine the short-term variability of plasma HIV-1 RNA level during stable therapy; the relative importance of pretreatment values and early changes in CD4+ count, HIV-1 RNA levels, and infectious HIV-1 titers in mononuclear cells of peripheral blood and pretreatment syncytium-inducing phenotype of an HIV-1 isolate for prediction of disease progression and decline in CD4+ counts during therapy. DESIGN: Data were collected prospectively in a randomized, clinical trial comparing two combination regimens (ACTG [AIDS Clinical Trials Group] Protocol 241) and pooled across treatments. SETTING: 8 AIDS Clinical Trials Units. PATIENTS: 198 adults with HIV-1 infection and no more than 350 CD4+ lymphocytes/mm3 who had received at least 6 months of nucleoside therapy. INTERVENTIONS: All patients received zidovudine and didanosine; 100 received nevirapine and 98 received placebo. MEASUREMENTS: CD4+ lymphocyte counts, plasma HIV-1 RNA levels, and infectious HIV-1 titers in cells were measured before and 8 and 48 weeks after study treatment. Assay for the syncytium-inducing viral phenotype was done at baseline. Progression was defined as occurrence of opportunistic infection, malignancy, or death during the 48 weeks after treatment began. RESULTS: The difference between two measurements of HIV-1 RNA levels at baseline was within +/-0.39 log10 copies/mL (2.5-fold) for 90% of 167 patients receiving stable therapy. In a multivariate model, risk for disease progression was reduced by 56% (95% CI, 8% to 79% [P = 0.028]) for every 10-fold lower HIV-1 RNA level at baseline, by 52% (CI, 6% increase to 79% reduction [P = 0.071]) for every 10-fold reduction in HIV-1 RNA level at 8 weeks after treatment initiation, and by 67% (CI, 42% to 81% [P < 0.001]) for every 2-fold higher CD4+ count at baseline. These risk factors and syncytium-inducing viral phenotype at baseline, but not infectious HIV-1 titers in circulating cells, were associated with change in CD4+ counts over 48 weeks. CONCLUSIONS: For an individual patient, a change in plasma HIV-1 RNA level of 2.5-fold or more probably indicates a true biological change. Monitoring HIV-1 RNA levels and CD4+ lymphocytes before a change in antiretroviral treatment and monitoring HIV-1 RNA levels shortly thereafter improves prediction of disease progression and decline in CD4+ counts for 1 year compared with monitoring CD4+ counts of HIV-1 RNA levels alone. Additional monitoring of infectious HIV-1 titers in mononuclear cells of peripheral blood is not useful.

A model system for human cytomegalovirus-mediated modulation of human immunodeficiency virus type 1 long terminal repeat activity in brain cells.

Previously, our laboratory showed that human cytomegalovirus (HCMV) activates human immunodeficiency virus type 1 (HIV-1) in brain-derived cells with limited HIV-1 gene expression but inhibits HIV-1 in cells fully permissive for replication of both viruses (F. M. Jault, S. A. Spector, and D. H. Spector, J. Virol. 68:959-973, 1994). To investigate these effects further, we developed a model system that uncouples HIV-1 gene expression from long terminal repeat (LTR) activity. Two monoclonal U373-MG astrocytoma/glioblastoma cell lines (LTRIG and LIGHIVDC) were generated, each containing an integrated copy of an LTR-chloramphenicol acetyltransferase (CAT) construct and the Escherichia coli lacI gene. LIGHIVDC also has an inducible HIV-1 genome controlled by a Rous sarcoma virus promoter with lac operator sequences. Basal LTR-mediated CAT activity is 65-fold higher in LIGHIVDC than in LTRIG, and this activity is further increased (20-fold) following incubation of LIGHIVDC with isopropyl-beta-D-thiogalactopyranoside (IPTG). Tat protein can be detected by immunostaining in LIGHIVDC. However, Rev-mediated transport and subsequent translation of the singly spliced and unspliced HIV-1 mRNAs is inefficient. In the absence of Tat, HCMV stimulated CAT activity approximately 20-fold, and this activation required HCMV gene expression but not viral DNA replication. LTR-directed transcription was unaffected by HCMV infection in LIGHIVDC but was inhibited in these cells when they contained increased Tat levels following IPTG induction. These results support the hypothesis that HCMV can induce the HIV-1 LTR when HIV-1 gene expression is minimal and that a threshold level of HIV-1 gene products is necessary for HCMV to inhibit this promoter.

Electrophysiologic and histologic studies in clinically unaffected muscles of patients with prior paralytic poliomyelitis.

Macro-electromyography (macro-EMG) studies have provided important information about the size of the motor units and the degree of reinnvervation in clinically affected muscles of patients with a history of poliomyelitis and postpolio syndrome. The study of clinically unaffected muscles and correlation of their electrophysiologic characteristics with the muscle architecture could provide meaningful information about the ongoing subclinical denervation. We performed macro-EMG and concomitantly measured fiber density in the clinically unaffected gastrocnemius muscle of 10 patients with postpolio syndrome and 10 normal subjects of similar age. We also performed biopsies on the gastrocnemius muscle of 8 of the patients. The median amplitude and area of the macro-motor unit potentials (macro-MUPs) were increased in 8 of the 10 patients, and occasionally were five times as large as the mean median value for the normal subjects. Seven biopsy specimens showed moderate to very large fiber-type grouping. In 5 patients, there was correlation between the electrophysiologic and histologic indices of reinnervation. Amplitude and area of the macro-MUPs were associated with the muscle fiber cross-sectional area. We conclude that clinically unaffected muscles of patients with postpolio syndrome often have large motor units as the result of effective reinnervation after the original motor neuron loss. In spite of possible differences in the cytoarchitecture of muscles affected to different degrees, macro-EMG and fiber density measurements are reliable noninvasive techniques for studying the extent and effectiveness of reinnervation in patients with postpolio syndrome.

Strength gains without muscle injury after strength training in patients with postpolio muscular atrophy.

We evaluated changes in the dynamic and isometric strength in the newly weakened quadriceps muscles and asymptomatic triceps muscles of 6 patients with postpolio muscular atrophy (PPMA) after 10 weeks of progressive resistance strength training. Alterations in muscle size were determined with magnetic resonance imaging. Serum creatine kinase levels were measured throughout training, and histological signs of muscle injury and changes in muscle fiber size and types were assessed with muscle biopsies before and after training. Exercise training led to an increase in dynamic strength of 41% and 61% for the two knee extensor tests, and 54% and 71% for the two elbow extensor tests. Up to 20% of the improvement was maintained 5 months after cessation of training. Isometric strength, whole muscle cross-sectional areas of quadriceps and triceps muscles, and serum muscle enzymes did not change. No destructive histopathological changes were noted in the repeat muscle biopsies, and no consistent changes in muscle fiber size or fiber type percentages were observed. These results demonstrate that a supervised resistance training program can lead to significant gains in dynamic strength of both symptomatic and asymptomatic muscles of PPMA patients without serological or histological evidence of muscular damage.

Replication of macrophage-tropic and T-cell-tropic strains of human immunodeficiency virus type 1 is augmented by macrophage-endothelial cell contact.

Macrophages perform a central role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection and have been implicated as the cell type most prominent in the development of central nervous system impairment. In this study, we evaluated the effect of interaction between macrophages and endothelial cells on HIV-1 replication. Upregulation of HIV-1 replication was consistently observed in monocyte-derived macrophages (hereafter called macrophages) cocultured with either umbilical vein endothelial cells or brain microvascular endothelial cells. HIV-1 p24 antigen production of laboratory-adapted strains and patient-derived isolates was increased 2- to 1,000-fold in macrophage-endothelial cocultures, with little or no detectable replication in cultures containing endothelial cells only. The upregulation of HIV-1 in macrophage-endothelial cocultures was observed not only for viruses with the non-syncytium-inducing, macrophage-tropic phenotype but also for viruses previously characterized as syncytium inducing and T-cell tropic. In contrast, cocultures of macrophages with glioblastoma, astrocytoma, cortical neuronal, fibroblast, and placental cells failed to increase HIV-1 replication. Enhancement of HIV-1 replication in macrophage-endothelial cocultures required cell-to-cell contact; conditioned media from endothelial cells or macrophage-endothelial cocultures failed to augment HIV-1 replication in macrophages. Additionally, antibody to leukocyte function-associated antigen (LFA-1), a macrophage-endothelial cell adhesion molecule, inhibited the enhanced HIV-1 replication in macrophage-endothelial cell cocultures. Thus, these data indicate that macrophage-endothelial cell contact enhances HIV-1 replication in macrophages for both macrophage-tropic and previously characterized T-cell-tropic strains and that antibody against LFA-1 can block the necessary cell-to-cell interaction required for the observed upregulation. These findings may have important implications for understanding the ability of HIV-1 to replicate efficiently in tissue macrophages, including those in the brain and at the blood-brain barrier.

Differential effects of human cytomegalovirus on integrated and unintegrated human immunodeficiency virus sequences.

Human cytomegalovirus (HCMV) has been implicated as a potential cofactor in human immunodeficiency virus type 1 (HIV-1)-related disease. Previously, we reported that HCMV inhibits HIV-1 RNA and protein synthesis in cells productively infected with both viruses but, in transient assays, activates an HIV-1 long terminal repeat-chloramphenicol acetyltransferase (LTR-CAT) construct introduced into the cell by transfection (V. Koval, C. Clark, M. Vaishnav, S. A. Spector, and D. H. Spector, J. Virol. 65:6969-6978, 1991). We show here that HCMV can also activate an infectious proviral HIV-1 genome transiently transfected into a cell. To ascertain whether integration of the HIV-1 provirus plays a role in these differential effects, we generated monoclonal and polyclonal cell lines that each contain a single integrated copy of an HIV-1 LTR-CAT construct and compared the regulatory effects of HCMV and HIV-1 infection in these cells with those occurring in the same type of cell transiently transfected with the HIV-1 LTR-CAT construct. We find that HCMV activates the transfected HIV-1 promoter 230-fold but activates the integrated promoter only 2.8- to 54-fold. In contrast, HIV-1 stimulates the integrated HIV-1 promoter 2,700- to 6,000-fold but stimulates the transfected promoter only 80-fold. Thus, the relative response of the HIV-1 promoter to HCMV and HIV-1 regulatory proteins depends upon whether it is integrated. To determine if HIV-1 gene products are necessary for the HCMV-mediated repression, we constructed cell lines containing two different stably integrated HIV-1 proviruses: one is tat- and nef-minus and transcriptionally inactive, while the other is env- and nef-minus but actively expresses the other HIV-1 gene products. Upon infection with HCMV, HIV-1 antigen production was stimulated from the inactive HIV-1 genome but inhibited from the active genome. We propose that HCMV has two separate effects on HIV-1 replication during a coinfection. One is a slight stimulatory effect which would be undetectable during an active HIV-1 infection, while the other is a net inhibitory effect that is mediated by an interaction between HCMV and HIV-1 gene products.

Human cord blood mononuclear cells are preferentially infected by non-syncytium-inducing, macrophage-tropic human immunodeficiency virus type 1 isolates.

Identification of the factors which impact on the transmission of human immunodeficiency virus type 1 (HIV-1) from an infected mother to her infant is essential for the development of effective strategies to prevent perinatal HIV-1 infection. The current study was designed to determine if unstimulated human neonatal cord blood mononuclear cells (CBMC) differ from adult peripheral blood mononuclear cells (PBMC) in susceptibility to HIV-1 infection. Both cell populations were challenged with two laboratory and two clinical HIV-1 isolates with different phenotypic properties. Infection was evaluated by quantitation of p24 antigen production and p24 antigen expression by an enzyme immunoassay and immunofluorescence, respectively. T-cell markers were determined by flow cytometry. Unstimulated CBMC were preferentially infected by macrophage-tropic, non-syncytium-inducing (non-SI) laboratory and clinical isolates, whereas PBMC were more susceptible to T-lymphotropic, SI HIV-1 strains. The macrophage-tropic strain HIV-1Ba-L replicated to 100-fold higher titers in CBMC than a similar inoculum of the SI isolate HIV-1LAI. The opposite occurred in unstimulated PBMC, which replicated HIVLAI to eightfold higher titers than the macrophage-tropic isolate. These findings indicate that a selection of viral phenotype may occur with unstimulated CBMC displaying a predominant susceptibility to infection by macrophage-tropic, non-SI HIV-1 strains and that this selection may influence mother-infant transmission of HIV-1.

Dysregulation of cytokine expression in monocytes from HIV-positive individuals.

Because HIV may alter the production of inflammatory factors produced by monocytes, the expression of tumor necrosis factor alpha (TNF-alpha), tissue factor (TF), interleukin (IL)-1 beta, and IL-6 was evaluated in 47 HIV-seropositive persons and seronegative control subjects. RNA was extracted from freshly isolated lipopolysaccharide (LPS)-stimulated or unstimulated monocytes. Cytokine and TF expression was quantitated by dot blot hybridization or a reverse transcription polymerase chain reaction (RT-PCR). A significant depression of TF mRNA was observed in LPS-stimulated monocytes (66% less in AIDS, 20% less in AIDS-related complex (ARC), and 0% less in asymptomatic patients), whereas normal responses were observed for TNF-alpha, IL-1 beta, and IL-6. When constitutive expression was measured in unstimulated monocytes by RT-PCR, a differential pattern was also observed. TNF-alpha and IL-1 beta were positive in 85% of asymptomatic persons, compared with only 27% of ARC and 42% of AIDS patients. Expression of IL-6 was observed in lower proportions, 27-30%, with no significant differences among disease states. All samples were negative for TF. Thus, the regulation of inflammatory molecules is differentially altered in individuals with HIV infection. TF is preferentially down-regulated, compared with TNF-alpha, IL-1 beta, and IL-6, in LPS-stimulated monocytes as patients progress to AIDS. TNF-alpha and IL-1 beta are preferentially up-regulated, compared with IL-6 and TF, in unstimulated monocytes in asymptomatic persons, with a loss of up-regulation as patients progress to AIDS.

Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy.

Drug susceptibility and mutations in the reverse transcriptase (RT) gene were analyzed with 167 virus isolates from 38 patients treated with nevirapine, a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) RT. Resistant isolates emerged quickly and uniformly in all patients administered nevirapine either as monotherapy or in combination with zidovudine (AZT). Resistance developed as early as 1 week, indicating rapid turnover of the virus population. The development of resistance was associated with the loss of antiviral drug activity as measured by CD4 lymphocyte counts and levels of HIV p24 antigen and RNA in serum. In addition to mutations at amino acid residues 103, 106, and 181 that had been identified by selection in cell culture, mutations at residues 108, 188, and 190 were also found in the patient isolates. Sequences from patient clones documented cocirculating mixtures of populations of different mutants. The most common mutation with monotherapy, tyrosine to cysteine at residue 181, was prevented from emerging by coadministration of AZT, which resulted in the selection of alternative mutations. The observations documented that, under selective drug pressure, the circulating virus population can change rapidly, and many alternative mutants can emerge, often in complex mixtures. The addition of a second RT inhibitor, AZT, significantly altered the pattern of mutations in the circulating population of HIV.