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Study patients had been treated with the three-drug regimen of AZT, 3TC, and IDV for a period of about 2 years (1) and were chosen for the study because, with the exception of sporadic detection of HIV RNA in the first year, they had plasma and serum HIV RNA concentrations at or below 50 copies/ml shortly after initiation of therapy, which persisted through the time point when the coculture was performed. All patients had been treated with AZT in the past, before enrollment in the Merck 035 study. All patients gave written informed consent, and the conduct of this study conforms to and was approved by the local institutional review board.
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note
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HIV RNA quantitation was performed with the Amplicor assay (Roche Molecular Systems) on cell-free virus supernatants from cocultures. Plasma and serum RNA concentrations were determined using the Ultrasensitive assay (Roche), a modification of the Amplicor assay (22). Briefly, this assay incorporates a high-speed spin (23,500g for 1 hour at 4°C) and resuspensbn of the RNA pellet in one-fourth of the typical diluent volume before the reverse transcription polymerase chain reaction (RT-PCR) and improves the sensitivity of detection from 400 copies/ ml to 50 copies/ml. Repeat assays were performed on appropriate dilutions of supernatants when results from standard Amplicor assays exceeded the upper limit of reliability for the assay. HIV p24 antigen was measured by enzyme-linked immunosorbent assay (ELISA; Coulter, Hialeah, FL).
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6 CD8-depleted/CD4-enriched cells used for each of the enhanced coculture conditions).
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data not shown
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Wong, J.K.1
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15144350527
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note
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In six of the seven individual coculture experiments, culture conditions incorporating either TNF-α or autologous macrophages or both (in addition to CD8 depletion and activation with CD3 and CD28 mAbs) resulted in the earliest and highest measured RNA concentrations (and p24 concentrations) by days 14 to 21. This occurred in two experiments with TNF-α alone, in three with macrophages alone, and in one with both TNF-α and macrophages.
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15144346353
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RNA extractions from plasma and viral supernatants were performed using the guanidinium isothiocyanate-phenol system provided with the Amplicor Monitor (Roche Molecular Systems, Branchberg, NJ). Population sequencing was performed with the GeneChip system (Affymetrix, Santa Clara, CA) (24) with the following modifications of the RT and PCR procedures as described in the current protocol (Rev. 3, April 1997): Primers used were PRT T7 (5′-GTAATACGACTCACTATAGGGCCACTAACTTCTGTATGTCATTGACAGTCCA-3′) for reverse transcription, PRT T3 (5′-AATTAACCCTCACTAAAGGGCAGACCAGAGCCAACAGCCCCA-3′) for PCR sense primer, and PRT T7 for antisense primer (T7 RNA and T3 RNA promoter sequences, respectively, are in italics). Reaction conditions were according to the manufacturer, using AMV RT (Gibco-BRL) for reverse transcription and rTth XL polymerase (Perkin-Elmer) for PCR amplification. The labeled, transcribed RNA was fragmented and then hybridized per manufacturer's specifications, to oligonucleotide probes arrayed on the GeneChip. GeneChips were scanned using a confocal laser microscope. The GeneChip 2.0 software (Affymetrix) with the Rules algorithm was used for base-calling and analysis of sequence data. The region sequenced includes all of the HIV protease region and the first 242 codons of the HIV RT. In all cases, verification of sequence data by the GeneChip method involving the known resistance-conferring codon was done by automated dideoxynucteotide sequencing with an ABI 373A sequencer (Applied Biosystems). Sequences have been submitted to GenBank (accession numbers AF027708 to AF027721). Phylogenetic analysis with tree-building based on maximum likelihood analysis was performed on the complete nucleotide sequence data sets with DNAML from Phylip 3.5 (25) and trees were produced using the DRAWTREE utility as described (25). Separate analysis using parsimony analysis with bootstrapping (×100) performed with SEQBOOT, DNAPARS, and CONSENSE, all in Phylip, gave similar results (18). Sequences for laboratory strains of HIV used in the comparative analysis were obtained electronically from NLBI/GenBank.
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We thank the patients at the San Diego AIDS Treatment Center who participated in this study; K. Nuffer, B. Coon, and G. Dyak for blood collection and patient recruitment; L. Terry, N. Keating, S. Albanil, V. Alvarez, and J. Aufderheide for technical assistance; E. Emini, J. Sninsky, R. Kornbluth, A. J. Leigh-Brown, and N. Riggs for their cooperation and advice; J. Guatelli for advice and critical review of the manuscript; K.-E. Hellstrom for CD28 mAb clone 9.3; and D. Smith and S. Wilcox for administrative assistance. Supported by National Institute of Allergy and Infectious Diseases grants Al 01361 (J.K.W.), Al 27670 (D.D.R.), Al 38858 (D.D.R.), Al 36214 (D.D.R.), and Al 29164 (Center for AIDS Research, San Diego); the Research Center for AIDS and HIV Infection of the San Diego Veterans Affairs Medical Center (D.D.R.); a Merit Award of the Department of Veterans Affairs (C.A.S.); and Swiss National Science Foundation grants 823A-050434 (M.H.) and 84AD-046176 (H.F.G.).
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