Chemokine receptor CCR5 Delta 32 genetic analysis using multiple specimen types and the NucliSens Basic Kit.

Resistance to HIV-1 infection and delayed disease progression have been associated with a 32-bp deletion (Delta32) in the gene encoding the CCR5 chemokine receptor. In the present study we describe the modification of a nucleic acid sequence-based amplification (NASBA)-based CCR5 genotyping assay for a NucliSens Basic Kit (Organon Teknika, Durham, N.C.) format using a new target-specific sandwich oligonucleotide detection methodology. The new method permitted the use of generic electrochemiluminescent probes supplied in the NucliSens Basic Kit, whereas the original NASBA method required expensive target-specific ruthenium detection probes. The Basic Kit CCR5 Delta32 genotypic analysis was in 100% concordance with both the original NASBA assay and DNA PCR results. This study also evaluated the use of multiple specimen types, including peripheral blood mononuclear cells (PBMC), whole blood, dried blood spots, buccal scrapings, and plasma, for CCR5 genotype analysis. The sensitivities of the three assays were comparable when PBMC or whole blood was the specimen source. In contrast, when dried blood spots, buccal scrapings, or plasma was used as the sample source, the sensitivity of DNA PCR was 80.95, 42.8, or 0%, respectively, compared to 100% sensitivity obtained with the original NASBA and Basic Kit NASBA assays. Our study indicates that the NucliSens Basic Kit NASBA assay is very sensitive and specific for CCR5 Delta32 genotyping using multiple sample types.

Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay.

Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

RANTES and MIP-1beta mRNA expression in human peripheral blood mononuclear cells: transcript quantification using NASBA technology.

The importance of chemokines in the immune response, as well as in a range of specific disease states, is becoming increasingly apparent. The role of CC- (or beta-) chemokines and their receptors in the pathology and mechanisms of HIV-1 infection has served to intensify interest in these factors. Although the functionality of these factors resides in their protein forms, assays for the detection and quantification of these protein factors in clinical samples are not readily available. Consequently, we designed NASBA-based assays for the quantification of the mRNA encoding two members of the CC-chemokine family: RANTES and MIP-1beta. The NASBA-based assays are extremely sensitive, accurate, and reproducible across a dynamic range of at least four orders of magnitude. Inter-assay performance is comparable to intra-assay performance. We applied these methods to the analysis of normal human PBMC and PBMC from HIV-1 infected individuals. Although MIP-1beta mRNA levels are higher than RANTES levels in both populations, RANTES levels in HIV-1+ patients are higher than in normal individuals. The utility of these assays in longitudinal studies of specific subpopulations of cells, as well as their potential use in clinical diagnostics, is discussed.

Macrophage-derived chemokine gene expression in human and macaque cells: mRNA quantification using NASBA technology.

Macrophage-derived chemokine (MDC) is a CC-chemokine that inhibits infection by both macrophage- and T cell-tropic strains of HIV-1. This suppressor activity has led to great interest in fully characterizing the role of MDC in the pathogenesis of HIV-1 infection. Methods for the quantitation of constitutive levels of MDC protein in vivo are lacking. In this report, we describe the development and performance of a NASBA-based assay for the quantification of MDC mRNA expression in human and macaque cells. Although the constitutive in vivo levels of MDC mRNA in macaque and human T lymphocytes were low, in vitro activation of these cells greatly increased MDC transcription. Levels in the human and macaque cells were comparable under all conditions tested. Positive correlations between MDC transcription and protein expression were observed. The results indicate that this assay is extremely sensitive and reproducible over a five log dynamic range, and effectively quantifies MDC mRNA in resting and activated T cells. This assay may therefore permit characterization of the role of MDC in HIV-1/SIV pathogenesis, and in vaccine-induced immune responses.

Quantitative evaluation of simian immunodeficiency virus infection using NASBA technology.

The most commonly used animal model for the study of HIV-1 infection in humans is the infection of non-human primates by simian immunodeficiency virus (SIV). The animal hosts used most frequently are different species of macaques, which are readily infected with SIV, and can therefore be used to study natural infection, pathogenesis, therapy, and vaccine efficacy. The study of HIV-1 infection in humans relies heavily on the quantification of HIV-1 load (i.e. viral RNA) in patient plasma. Given the importance of HIV-1 RNA levels in humans, it follows that SIV RNA levels in animals are also relevant to the study of infection in this model system. This report describes the development of the isothermal amplification-based NASBA technology for the quantification of SIV RNA load in macaque plasma. Evaluation of the assay using model systems demonstrated that the assay is accurate and reproducible over nearly four orders of magnitude. Viral RNA load data were compared to other infection measurements in the macaque system. Further, the assay was used to provide copy number levels of SIV RNA in macaque plasma samples, permitting characterization of viral load during the course of SIV infection.

Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization.

The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

Coinfection of macaques with simian immunodeficiency virus and simian T cell leukemia virus type I: effects on virus burdens and disease progression.

To test the hypothesis that coinfection with human immunodeficiency virus (HIV) and human T cell leukemia/lymphoma virus types I or II (HTLV-I or -II) accelerates progression to AIDS, pig-tailed macaques were inoculated with the simian counterparts, SIV and STLV-I. During 2 years of follow-up of singly and dually infected macaques, no differences in SIV burdens, onset of disease, or survival were detected. However, in the first coinfected macaque that died of AIDS (1 year after infection), >50% of CD4+ and CD8+ lymphocytes expressed CD25. On the basis of the low incidence of HTLV-I- and STLV-I-associated disease during natural infections, this early evidence of neoplastic disease was unexpected. While these results demonstrate that coinfection with SIV and STLV-I has no influence on the development of immunodeficiency disease, they do establish a reliable macaque model of persistent STLV-I infection.

Natural killer cells from human immunodeficiency virus (HIV)-infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro.

Macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell expressed and secreted), which are the natural ligands of the CC-chemokine receptor CCR5, inhibit replication of MT-2- negative strains of HIV-1 by interfering with the ability of these strains to utilize CCR5 as a coreceptor for entry in CD4(+) cells. The present study investigates the capacity of natural killer (NK) cells isolated from HIV-infected individuals to produce CC-chemokines and to suppress HIV replication in autologous, endogenously infected cells as well as to block entry of MT-2-negative HIV into the CD4(+) T cell line PM-1. NK cells freshly isolated from HIV-infected individuals had a high number of mRNA copies for MIP-1alpha and RANTES. NK cells produced significant amounts of RANTES, MIP-1alpha, and MIP-1beta constitutively, in response to stimulation with IL-2 alone and when they were performing their characteristic lytic activity (K562 killing). After CD16 cross-linking and stimulation with IL-2 or IL-15 NK cells produced CC-chemokines to levels comparable to those produced by anti-CD3-stimulated CD8(+) T cells. Furthermore, CD16 cross-linked NK cells suppressed (49-97%) viral replication in cocultures of autologous CD8/NK-depleted PBMC to a degree similar to that of PHA or anti-CD3-stimulated CD8(+) T cells. In 50% of patients tested, NK-mediated HIV suppression could be abrogated by neutralizing antibodies to MIP-1alpha, MIP-1beta and RANTES; in contrast, CD8(+) T cell-mediated suppression was not significantly overcome upon neutralization of CC-chemokines. Supernatants derived from cultures of CD16 cross-linked NK cells stimulated with IL-2 or IL-15 dramatically inhibited entry of a MT-2-negative strain of HIV, BaL, in the CD4(+)CCR5(+) PM-1 T cell line. These data suggest that activated NK cells may be an important source of CC-chemokines in vivo and may suppress HIV replication by CC-chemokine-mediated mechanisms in addition to classic NK-mediated lytic mechanisms.

Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma.

To define the optimal blood collection parameters for plasma human immunodeficiency virus type 1 (HIV-1) viral load testing, plasma HIV-1 RNA levels were quantitated with the NASBA HIV-1 RNA QT System from blood specimens that were collected, processed, and stored under a variety of conditions that might have affected HIV-1 RNA stability. We determined that when whole blood was processed within 2 h of specimen collection the levels of HIV-1 RNA detected in EDTA-, heparin-, and acid citrate dextrose (ACD)-anticoagulated plasma samples were comparable. The levels of HIV-1 RNA in serum specimens (mean = 4.126 log units) were significantly lower (P < 0.01) than the levels in corresponding plasma samples (mean = 4.501 log units). One cycle of freeze-thaw (-70 degrees C) did not significantly reduce the level of HIV-1 RNA detected in EDTA-, heparin-, or ACD-anticoagulated plasmas. The EDTA-anticoagulated plasmas showed the smallest decrease in HIV-1 RNA copies (0.050 log units). HIV-1 RNA levels decreased over a 6-month time period in serum as well as in EDTA-, ACD-, and heparin-anticoagulated plasmas stored at -70 degrees C. However, the only significant decreases were for serum (mean decrease = 0.317 log units) and heparin-anticoagulated samples (mean decrease = 0.384 log units). A comparison of the levels of HIV-1 RNA in cell-free plasma collected in VACUTAINER EDTA Plasma Preparation Tubes and in standard VACUTAINER EDTA tubes determined that HIV-1 RNA levels were stable for up to 30 h after collection when stored at either room temperature (mean standard deviation [SD] = +/- 0.101 log units) or at 4 degrees C (mean SD = +/- 0.102 log units) as cell-free plasma or as EDTA-anticoagulated whole blood (mean SD = +/- 0.109 log units). These data indicate that EDTA-anticoagulated plasma is the most suitable and stable matrix for HIV-1 RNA quantitation.

NASBA technology: isothermal RNA amplification in qualitative and quantitative diagnostics.

Nucleic acid amplification technologies allow for the development of highly sensitive and specific diagnostic assays. The capacity to amplify and detect analyte targets, which may be present in a clinical sample as a single copy; is characteristic of many of these amplification technologies. NASBA is an isothermal method of nucleic acid amplification with such capability, and is particularly well suited for the amplification of RNA analytes. NASBA utilizes the coordinated activities of three enzymes (AMV-RT, RNase H, T7 RNA polymerase), and two oligonucleotide primers which are specific for the analyte target. The amplification process is part of a total system which includes a versatile nucleic acid isolation procedure, and powerful detection methodology. In this report, the development of NASBA technology for the detection of human Retrovirus RNA will be discussed. Specifically, a qualitative NASBA assay for the RNA of HTLV I, and a quantitative NASBA assay for HIV-1 will be described.

NASBA: a novel, isothermal detection technology for qualitative and quantitative HIV-1 RNA measurements.

Although immunoassays have long served as the standard in the field of diagnostics, the advent of nucleic acid amplification technologies allows for a new array of diagnostic applications. NASBA, nucleic acid sequence-based amplification, is one such technology that is highly suited for the amplification of RNA. As such, NASBA is applied readily as a diagnostic tool for infectious diseases, particularly for RNA viruses, such as retroviruses. The development and application of NASBA technology as a qualitative and quantitative diagnostic system for HIV-1 are described in this article.

Detection of HIV-1 infection in vitro using NASBA: an isothermal RNA amplification technique.

Establishment of a sensitive infection assay for HIV-1 is essential for successful screening of antiviral agents and neutralizing antibodies. In this report, an infection assay is described which measures the expression of viral genomic RNA and spliced mRNA intermediates in infected cells by an amplification-based technique called NASBA. The extreme sensitivity of this method permits the detection of viral RNA in peripheral blood mononuclear cells (PBMC) within 48 h of infection by a low dose of virus. Similarly, spliced HIV-1 mRNA could be detected within 24 h of infection of CEM cells by HIV-1IIIB. This NASBA-based infection assay was shown to titer the neutralization of the HIV-1IIIB isolate by serum from an infected human and by a monoclonal antibody to gp120. Furthermore, the inhibitory effects of azidothymidine (AZT) and soluble CD4 on HIV-1IIIB infection were quantitated by this assay. The early detection of virus by NASBA minimizes the contribution of secondary infection, thereby permitting more accurate evaluation of antiviral agents and neutralizing antibodies. This assay may be useful for the study of infection of phenotypically distinct HIV-1 isolates, which differ in terms of their replication kinetics.

p53 mutations, ras mutations, and p53-heat shock 70 protein complexes in human lung carcinoma cell lines.

The p53 tumor suppressor gene is frequently mutated and the K-ras oncogene is occasionally mutated in primary specimens of human lung carcinomas. These mutated genes also cooperate in the immortalization and neoplastic transformation of rodent cells. To determine whether these mutations are necessary for maintenance of the immortalized and/or neoplastically transformed states of human bronchial epithelial cells, the p53 gene and regions of the ras (K-, H-, and N-) genes were sequenced in nine human lung carcinoma cell lines. Detection of p53 mutations by polymerase chain amplification and direct DNA sequencing was corroborated by p53 immunocytochemistry and coimmunoprecipitation of p53 with heat shock protein 70. p53 and ras genes were frequently, but not always, mutated in the carcinoma cell lines. These data are consistent with the hypothesis that multiple genetic changes involving both protooncogenes and tumor suppressor genes occur during lung carcinogenesis.

Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53.

To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation we constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastoma multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. We show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits G0/G1 progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the G0/G1 phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.

Wild type human p53 is antiproliferative in SV40-transformed hamster cells.

The transformation related protein p53 has been implicated in the process of normal cell proliferation and neoplastic transformation. In this study, the influence of wild type human p53 on cell proliferation was examined. Plasmid constructs encoding the wild type human p53 and various mutant p53 cDNAs, driven by the mouse mammary tumor virus (MMTV) promoter linked to the dominant biochemical selection marker gpt, were used in a colony forming assay employing SV40 transformed HR8 hamster cells. Plasmids encoding wild type p53 drastically reduced the number of gpt+ colonies obtained after transfection, whereas the mutant forms of p53 had no effect. Stable clonal hamster cell lines that constitutively express wild type p53 were isolated and found to have altered growth characteristics (i.e. lower saturation densities, increased doubling times). These findings are consistent with the notion that wild type p53 protein could function as a growth suppressor. The potential role of p53 in the normal cell cycle and in the transformation process is discussed.

Identification and characterization of a p53 gene mutation in a human osteosarcoma cell line.

The nuclear phosphoprotein p53 occurs at elevated levels in many transformed cells. Mutant forms of mouse p53 possess enhanced transforming activity compared with wild type p53. Mutant mouse p53 proteins form complexes with the 70 kDa family of heat shock proteins (HSPs). We previously demonstrated an association between p53 and the 70 kDa HSPs in the human osteosarcoma (HOS) derived cell line HOS-SL. We report here the molecular cloning and sequencing of the p53 gene from HOS-SL cells, and demonstrate that it is in fact mutant. Further, analysis of similar HOS-derived cell lines demonstrates that they also encode the same mutant form of p53, whereas the wild type form of p53 appears to be lost in these cells. Stability studies demonstrate an increased half life of the p53 protein in these cells, in keeping with its association with the HSP 70 proteins. A potential role for this p53 mutant in the transformation process is discussed.