RESUMO
BACKGROUND: The majority of prostate cancers (CaP) are detected in early stages with uncertain prognosis. Therefore, an intensive effort is underway to define early predictive markers of CaP with aggressive progression characteristics. METHODS: In order to define such prognostic markers, we performed comparative analyses of transcriptomes of well- and poorly differentiated (PD) tumor cells from primary tumors of patients (N=40) with 78 months of mean follow-up after radical prostatectomy. Validation experiments were carried out at transcript level by quantitative real-time reverse transcriptase-PCR (RT-PCR) (N=110) and at protein level by immunohistochemistry (N=53) in primary tumors from an independent patient cohort. RESULTS: Association of a biochemical network of 12 genes with SPARC gene as a central node was highlighted with PD phenotype. Of note, there was remarkable enrichment of NKXH_NKXH_HOX composite regulatory elements in the promoter of the genes in this network suggesting a biological significance of this gene-expression regulatory mechanism in CaP progression. Further, quantitative expression analyses of SPARC mRNA in primary prostate tumor cells of 110 patients validated the association of SPARC expression with poor differentiation and higher Gleason score. Most significantly, higher SPARC protein expression at the time of prostatectomy was associated with the subsequent development of metastasis (P=0.0006, AUC=0.803). CONCLUSIONS: In summary, we propose that evaluation of SPARC in primary CaP has potential as a prognostic marker of metastatic progression.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Ósseas/secundário , Metástase Linfática/genética , Osteonectina/biossíntese , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologia , Diferenciação Celular , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Prostatectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/cirurgia , RNA Mensageiro/metabolismoRESUMO
Separations can be broadly categorized as preparative, where the objective is to extract purified quantities of a sample from a complex mixture, or analytic, where the goal is to determine and quantify the contents of the original mixture. Here we demonstrate the application of a new microfluidic separation method, isodielectric separation (IDS), to a range of analytic separations involving cells and particles spanning several orders of magnitude in volume and electrical conductivity. In IDS, cells are dielectrophoretically concentrated to the region along an electrical conductivity gradient where their polarizability vanishes; by measuring this position--the isodielectric point (IDP)--as operating conditions such as the frequency and voltage of the applied electric field are varied, we are able to sort cells or particles with distinct IDPs while simultaneously characterizing their electrical properties. We apply this technique to measure the electrical properties of polystyrene microspheres, viable and nonviable cells of the budding yeast Saccharomyces cerevisiae , and murine pro B cells, including how these electrical properties vary with the electrical conductivity of the surrounding solvent.
Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Técnicas Analíticas Microfluídicas/métodos , Animais , Linhagem Celular , Sobrevivência Celular , Impedância Elétrica , Temperatura Alta , Leucócitos/citologia , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Microesferas , Tamanho da Partícula , Poliestirenos/química , Poliestirenos/isolamento & purificação , Saccharomyces cerevisiae/citologiaRESUMO
The high prevalence of TMPRSS2-ERG rearrangements ( approximately 60%) in prostate cancer (CaP) leads to androgenic induction of the ETS-related gene (ERG) expression. However, the biological functions of ERG overexpression in CaP remain to be understood. ERG knockdown in TMPRSS2-ERG expressing CaP cells induced striking morphological changes and inhibited cell growth both in cell culture and SCID mice. Evaluation of the transcriptome and specific gene promoters in ERG siRNA-treated cells and investigation of gene expression signatures of human prostate tumors revealed ERG-mediated activation of C-MYC oncogene and the repression of prostate epithelial differentiation genes (PSA and SLC45A3/Prostein). Taken together, these data combining cell culture and animal models and human prostate tumors reveal that ERG overexpression in prostate tumor cells may contribute to the neoplastic process by activating C-MYC and by abrogating prostate epithelial differentiation as indicated by prostate epithelial specific markers.
Assuntos
Diferenciação Celular , Proteínas de Fusão Oncogênica/genética , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/prevenção & controle , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas , Próstata/metabolismo , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Separations represent a fundamental unit operation in biology and biotechnology. Commensurate with their importance is the diversity of methods that have been developed for performing them. One important class of separations are equilibrium gradient methods, wherein a medium with some type of spatial nonuniformity is combined with a force field to focus particles to equilibrium positions related to those particles' intrinsic properties. A second class of techniques that is nonequilibrium exploits labels to sort particles based upon their extrinsic properties. While equilibrium techniques such as iso-electric focusing (IEF) have become instrumental within analytical chemistry and proteomics, cell separations predominantly rely upon the second, label-based class of techniques, exemplified by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). To extend the equilibrium techniques available for separating cells, we demonstrate the first implementation of a new microfluidic equilibrium separation method, which we call isodielectric separation (IDS), for sorting cells based upon electrically distinguishable phenotypes. IDS is analogous to isoelectric focusing, except instead of separating amphoteric molecules in a pH gradient using electrophoresis, we separate cells and particles in an electrical conductivity gradient using dielectrophoresis. IDS leverages many of the advantages of microfluidics and equilibrium gradient separation methods to create a device that is continuous-flow, capable of parallel separations of multiple (>2) subpopulations from a heterogeneous background, and label-free. We demonstrate the separation of polystyrene beads based upon surface conductance as well as sorting nonviable from viable cells of the budding yeast Saccharomyces cerevisiae.
Assuntos
Eletroforese/métodos , Citometria de Fluxo/métodos , Tamanho da Partícula , Poliestirenos/química , Poliestirenos/isolamento & purificação , Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Formalin-fixed paraffin-embedded (FFPE) prostate specimens are rich sources of molecular pathological information. However, FFPE-based microarray analysis of tissue samples may be hampered by the degradation and chemical alteration of RNA molecules due to the preservation procedure. In this report, we employed a probe analyses of Affymetrix oligonucleotide arrays at individual probe level to compensate for the potential loss of gene identifications associated with compromised mRNA quality in FFPE preparations. Furthermore, to increase the sample quality, we utilized laser capture microdissection of prostate tumor and benign epithelial cells. Remarkably, combination of these approaches recapitulated the common prostate cancer-associated gene expression alteration. Identification of prostate cancer associated-gene expression alterations such as AMACR, Kallikrein gene family and genes associated with androgen signaling such as PDEF and STEAP were consistent with previous findings reported in prostate cancer. These data suggest that combination of laser capture dissection with computational enhancement of microarray data may be useful for the assessment of gene expression changes in FFPE prostate cancer specimens.
Assuntos
Adenocarcinoma/genética , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Proteínas de Neoplasias/genética , Próstata/metabolismo , Neoplasias da Próstata/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Preservação de Tecido/métodos , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Dissecação/instrumentação , Dissecação/métodos , Formaldeído , Humanos , Processamento de Imagem Assistida por Computador , Lasers , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Inclusão em Parafina , Prostatectomia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Estabilidade de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , RNA Neoplásico/biossíntese , RNA Neoplásico/isolamento & purificação , Fixação de TecidosRESUMO
Thai residents have a greater risk of heterosexual transmission of human immunodeficiency virus (HIV) than do US residents. To analyze host factors associated with heterosexual transmission, vaginal epithelial biopsies from HIV-seropositive Thai and US women were evaluated for tissue virus load and histologic makeup. In all, 84% of Thai and 14% of US women exhibited a chronic inflammatory T cell infiltrate in the vaginal epithelium. In Thai tissue, the infiltrate was associated with elevated levels of HIV RNA in the epidermis. Uninfected Thai women also had vaginal epithelial inflammation. Inflammation did not correlate with sexually transmitted diseases or HIV disease stage. The higher rates and increased risk of heterosexual transmission in Thailand may be due to chronic inflammation at the site where the virus is transmitted, which leads to the accumulation of activated T cells. Such cells might act as targets for initial viral infection and subsequently as reservoirs that support efficient transmission.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , RNA Viral/análise , Vagina/virologia , Vaginite/imunologia , Adulto , Contagem de Linfócito CD4 , Epitélio/imunologia , Epitélio/patologia , Epitélio/virologia , Feminino , Infecções por HIV/imunologia , Humanos , Células de Langerhans/imunologia , Pessoa de Meia-Idade , RNA Viral/sangue , Infecções Sexualmente Transmissíveis/diagnóstico , Tailândia , Estados Unidos , Vagina/imunologia , Vagina/patologia , Carga ViralRESUMO
We have evaluated two commercially available kits (AMPLICOR MONITOR [Roche] and NASBA HIV-1 QT or NucliSens HIV-1 QT [Organon Teknika]) and two noncommercial methods for the accurate quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in seminal plasma. The same panels of coded specimens were tested on four separate occasions. Laboratories using the commercial assays employed silica beads to isolate HIV-1 RNA, which removed inhibitory factors sometimes found in seminal plasma. Sensitivities and specificities, respectively, for each assay were as follows: AMPLICOR MONITOR, 100 and 73%; NASBA HIV-1 QT, 84 and 100%; NucliSens HIV-1 QT, 99 and 98%; and noncommercial assays, 91 and 73%. When results from the laboratory that was inexperienced with the silica bead extraction method were excluded from the analysis, specificity for the Roche assay increased to 100%. The commercial assays demonstrated highly reproducible results, with intra-assay standard deviations (measured in log(10) RNA copies/milliliter of seminal plasma) ranging from 0.11 to 0.32; those of the noncommercial assays ranged from 0.12 to 0.75. Differences in mean estimated HIV-1 RNA concentrations were =0.67 log(10) and were greater at low viral loads. Suspension matrices that used blood plasma or seminal plasma did not make a difference in recovery of HIV-1 RNA, which suggested that blood plasma specimens can be used as external controls for seminal plasma assays. More variation in the HIV-1 RNA viral loads was observed in the seminal plasma values than in the blood plasma values when paired specimens from HIV-1-infected men were tested. Quantitation of HIV-1 RNA in seminal plasma can be reliably accomplished using two commercially available assays, and may be incorporated into the evaluations of HIV-1 seropositive men enrolled in clinical studies.
Assuntos
Infecções por HIV/diagnóstico , HIV-1/isolamento & purificação , RNA Viral/análise , Kit de Reagentes para Diagnóstico , Sêmen/virologia , Análise de Variância , Estudos de Avaliação como Assunto , Infecções por HIV/sangue , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Carga ViralRESUMO
The advent of high-density gene array technology has revolutionized approaches to drug design, development, and characterization. At the laboratory level, the efficient, consistent, and dependable exploitation of this complex technology requires the stringent standardization of protocols and data analysis platforms. The Affymetrix YE6100 expression GeneChip platform was evaluated for its performance in the analysis of both global (6,000 yeast genes) and targeted (three pleiotropic multidrug resistance genes of the ATP binding cassette transporter family) gene expression in a heterologous yeast model system in the presence and absence of the antimalarial drug chloroquine. Critical to the generation of consistent data from this platform are issues involving the preparation of the specimen, use of appropriate controls, accurate assessment of experiment variance, strict adherence to optimized enzymatic and hybridization protocols, and use of sophisticated bioinformatics tools for data analysis.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Resistência Microbiana a Medicamentos/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Saccharomyces cerevisiae/genética , Transportadores de Cassetes de Ligação de ATP/genética , Algoritmos , Resistência a Múltiplos Medicamentos/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
BACKGROUND: Regular testing of military personnel identifies early HIV infection; this identification provides a sentinel cohort in which to describe the evolving molecular epidemiology of HIV-1 transmission. OBJECTIVE: To describe the prevalence and epidemiologic correlates associated with the acquisition of non-subtype B and drug-resistant HIV infections. DESIGN: Cross-sectional study. SETTING: Military referral hospital. PATIENTS: 95 military personnel with HIV-1 seroconversion. MEASUREMENTS: Self-reported questionnaire, CD4 cell counts, plasma HIV-1 RNA levels, and nucleic acid sequence analysis for drug-resistant mutations and HIV-1 genetic subtype. RESULTS: 95 patients were enrolled between February 1997 and February 1998. The likely geographic location of HIV-1 acquisition was overseas in 8% of patients, the United States in 68%, and either overseas or the United States in 24%. Seven patients (7.4%) had subtype E infection; the remainder had subtype B infection. Eight of 31 (26%) treatment-naive patients had mutations in the reverse transcriptase or protease gene associated with drug resistance. CONCLUSIONS: The percentage of HIV-1 non-subtype B infection and antiretroviral drug-resistant mutations was relatively high in U.S. military personnel with recently acquired HIV-1 infection.
Assuntos
Soropositividade para HIV/epidemiologia , Soropositividade para HIV/virologia , HIV-1/genética , Militares , Contagem de Linfócito CD4 , Estudos Transversais , Resistência Microbiana a Medicamentos/genética , Endopeptidases/genética , Feminino , Genótipo , Transcriptase Reversa do HIV/genética , Soropositividade para HIV/genética , HIV-1/isolamento & purificação , Humanos , Masculino , Mutação , RNA Viral/sangue , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
The performance of a silica chip-based resequencing method, the Affymetrix HIV PRT 440 assay (hereafter referred to as the Affymetrix assay), was evaluated on a panel of well-characterized nonclade B viral isolates and on isolates exhibiting length polymorphisms. Sequencing of human immunodeficiency virus type 1 (HIV-1) pol cDNAs from clades A, C, D, E, and F resulted in clade-specific regions of base-calling ambiguities in regions not known to be associated with resistance polymorphisms, as well as a small number of spurious resistance polymorphisms. The Affymetrix assay failed to detect the presence of additional serine codons distal to reverse transcriptase (RT) codon 68 that are associated with multinucleoside RT inhibitor resistance. The increasing prevalence of non-clade B HIV-1 strains in the United States and Europe and the identification of clinically relevant pol gene length polymorphisms will impact the generalizability of the Affymetrix assay, emphasizing the need to accommodate this expanding pool of pol genotypes in future assay versions.
Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , Bioensaio/métodos , Resistência Microbiana a Medicamentos/genética , Genoma Viral , HIV-1/genética , HIV-1/efeitos dos fármacos , Humanos , Técnicas Microbiológicas , Polimorfismo de Fragmento de Restrição , RNA Viral/análise , RNA Viral/genéticaRESUMO
The assessment of viral load in the blood, tissues, and bodily fluids of persons infected with human immunodeficiency virus is fundamental to defining the stage of disease (1-3), the effect of antiviral treatments to abate disease (4, 5), disease progression (6-8), and propensity for the transmission of disease (9,10) as well. Now accepted as a surrogate for all of these features of HIV-1 disease, specific guidelines have been adopted for the use of viral load measures in the clinical management of the disease (11).
RESUMO
The fundamental clinical, viral, and immunologic features of early-stage human immunodeficiency virus type 1 (HIV-1) disease were examined in a seroprevalent cohort of 28 men and 14 women assessed longitudinally at three equally dispersed time points over a mean of 43 months. There were no gender differences in the relative risk of developing AIDS-defining end points or death. The median serum RNA levels assessed at the three study time points were 3.3-, 4.9-, and 1.5-fold lower, respectively, in women than in men. This suggests that while serum virus load may be as powerful a correlate of disease status in women as it is in men, the absolute values of the virus levels may be different in the 2 populations. These observations may have implications for the interpretation of levels of virus burden in women for the assessment of disease progression, transmission, and treatment.
Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , HIV-1/isolamento & purificação , Viremia/virologia , Adulto , DNA Viral/sangue , Feminino , Humanos , Masculino , Fatores SexuaisRESUMO
A number of quantitative assays have been developed by using amplification techniques to measure human immunodeficiency virus type 1 RNA in the plasma of infected individuals. The Virology Committee of the AIDS Clinical Trials Group in the Division of AIDS, National Institute of Allergy and Infectious Diseases, has established a quality assurance program (QAP) for quantitative assays of HIV-1 RNA levels in plasma. The primary objective of the QAP was to ascertain that a laboratory could maintain the precision required to have a 90% power to detect a fivefold difference in RNA copy number between two samples in the same batch. To achieve this goal, the QAP required an intra-assay standard deviation of no greater than 0.15 log10 RNA copies per ml. Panels for proficiency testing consisted of coded replicate samples and a common set of standards. To date, 41 laboratories have participated in the program and have used both commercial and in-house assays. We demonstrated that 65% of the laboratories were capable of attaining the necessary level of intra-assay precision. The fitted regressions indicated that the differences among laboratories that used the same kit were generally greater than the differences among population-average regressions for the kits themselves. The use of an external QAP and a common set of standards reduced differences both among laboratories that used the same kit and among laboratories that used different kits. Thus, use of a common set of standards across clinical trial protocols would allow for cross-protocol comparisons.
Assuntos
HIV-1/isolamento & purificação , RNA Viral/sangue , RNA Viral/normas , Virologia/métodos , Virologia/normas , Protocolos Clínicos/normas , Ensaios Clínicos como Assunto/normas , Estudos de Avaliação como Assunto , HIV-1/genética , Humanos , Laboratórios , Estudos Multicêntricos como Assunto/normas , National Institutes of Health (U.S.) , Controle de Qualidade , RNA Viral/genética , Padrões de Referência , Estados UnidosRESUMO
The life cycle of human immunodeficiency virus type 1 (HIV-1) is critically dependent on the transregulatory proteins Tat and Rev. Tat increases the production of HIV-specific mRNAs by direct binding to the transactivation response (TAR) element located at the 5' end of all HIV transcripts. In contrast, Rev uses a complex RNA stem loop structure, the Rev response element (RRE), which is found in full-length and singly spliced HIV transcripts. Rev is required for the cytoplasmic expression of full-length mRNAs encoding Gag, Pol, and Env structural proteins. The complex intracellular interactions between Tat, Rev, host cell factors, and their respective RNA response elements should be susceptible to interdiction by genetic therapies designed to introduce and express novel genetic information. We show that the expression of antisense RREs inhibited the cytoplasmic expression of RRE containing HIV-1 transcripts. HIV-based retroviral vectors containing either the antisense (-) or sense (+) RREs inhibited HIV replication in transient transfections. The production of full-length HIV mRNA was also decreased significantly by the expression of RREs in either orientation. Interestingly, there was a paradoxic increase in HIV p24 gag production at low levels of inhibitor; this effect may have been the result of encapsidation of RRE-containing HIV-based retroviral vectors. The data suggest that the introduction and inducible expression of RRE-containing, HIV-based retroviral vectors may have therapeutic value in HIV infection.
Assuntos
Elementos Antissenso (Genética)/fisiologia , Genes env/fisiologia , Vetores Genéticos , HIV-1/fisiologia , Replicação Viral/genética , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/química , Regulação Viral da Expressão Gênica , Genes tat/fisiologia , HIV-1/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Testes de Precipitina , Splicing de RNA , RNA Mensageiro/biossíntese , Coelhos , TransfecçãoRESUMO
Because stimulation of CD4+ lymphocytes leads to activation of human immunodeficiency virus-type 1 (HIV-1) replication, viral spread, and cell death, adoptive CD4+ T cell therapy has not been possible. When antigen and CD28 receptors on cultured T cells were stimulated by monoclonal antibodies (mAbs) to CD3 and CD28 that had been immobilized, there was an increase in the number of polyclonal CD4+ T cells from HIV-infected donors. Activated cells predominantly secreted cytokines associated with T helper cell type 1 function. The HIV-1 viral load declined in the absence of antiretroviral agents. Moreover, CD28 stimulation of CD4+ T cells from uninfected donors rendered these cells highly resistant to HIV-1 infection. Immobilization of CD28 mAb was crucial to the development of HIV resistance, as cells stimulated with soluble CD28 mAb were highly susceptible to HIV infection. The CD28-mediated antiviral effect occurred early in the viral life cycle, before HIV-1 DNA integration. These data may facilitate immune reconstitution and gene therapy approaches in persons with HIV infection.
Assuntos
Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Ativação Linfocitária , Anticorpos Monoclonais/imunologia , Complexo CD3/imunologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/citologia , Divisão Celular , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Interleucina-2/farmacologia , Fito-Hemaglutininas/farmacologia , Integração Viral , Replicação ViralRESUMO
Human immunodeficiency virus (HIV)-1 DNA and RNA levels and T lymphocyte cell surface markers were measured in blood serum and cell fractions from asymptomatic infected patients to find novel virologic and immunologic features in early disease predictive of subsequent clinical disease course. Thirty-two patients with rapid disease progression (rapid CD4+ cell loss and progression to clinical AIDS) were compared with 25 patients with stable infections (constant or rising CD4+ cell counts, no clinical disease manifestations). All HIV-1 burdens measured by polymerase chain reaction were consistently higher in specimens from rapid progressors than slow progressors. For each patient, virus burden remained relatively constant throughout the study period (mean, 42-44 months). Flow cytometry also disclosed stable lymphocyte immunophenotype patterns that correlated strongly with subsequent rapid progression to clinical disease. Thus, in early HIV-1 infection, a constellation of high virus burden and in vivo costimulatory antigen and lymphocyte activation abnormalities is predictive of rapid disease course.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/microbiologia , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Sequência de Bases , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , Primers do DNA/química , DNA Viral/análise , Feminino , Proteína do Núcleo p24 do HIV/imunologia , Antígenos HLA-DR/análise , Humanos , Hipersensibilidade Tardia/imunologia , Memória Imunológica , Imunofenotipagem , Células Matadoras Naturais/imunologia , Masculino , Medicina Militar , Dados de Sequência Molecular , Estudos Prospectivos , RNA Viral/análise , Receptores de Antígenos de Linfócitos T gama-delta/análise , Fatores de TempoRESUMO
To better understand the biologic meaning and potential clinical utility of p24 antigen measurements in human immunodeficiency virus (HIV) infection, p24 antigen and antibody and HIV RNA were quantitated in parallel. Specimens (n = 311) were analyzed from 74 participants in a zidovudine treatment study. Parallel antigen and RNA measurements revealed the frequent occurrence of two types of discordant results. First, p24 antigen was often not detected in samples with high antibody levels even when > 10(6) RNA copies/mL were present. Second, in specimens in which p24 antigen was detected, the concentration was greater than expected on the basis of HIV RNA values. These results suggest that optimal use of serum p24 antigen values will require consideration of both specific antibody levels and non-virion associated antigen.
Assuntos
Anticorpos Anti-HIV/sangue , Proteína do Núcleo p24 do HIV/sangue , Infecções por HIV/tratamento farmacológico , RNA Viral/sangue , Zidovudina/uso terapêutico , Adulto , Sequência de Bases , Western Blotting , Contagem de Linfócito CD4 , Feminino , Infecções por HIV/imunologia , Humanos , Masculino , Dados de Sequência Molecular , Estatística como AssuntoRESUMO
The potential of lymph node fine-needle aspiration (LNFNA) for sampling viral load was evaluated in excised, peripheral lymph nodes from five patients with early-stage human immunodeficiency virus type 1 HIV-1 disease (asymptomatic, CD4 cells > 300/mm3). The preponderance (> 80%) of viral RNA was within follicular germinal centers as noted by in situ hybridization on lymph node frozen sections (LNFSs) as well as within cohesive groups of 10-20 lymphoid cells (microfragments) in LNFNA preparations. Quantification of cells expressing HIV-1 RNA by in situ hybridization, quantification of HIV-1 gag RNA and gag DNA per 10(5) cells by polymerase chain reaction, and measurement of p24 antigen per 10(5) cells yielded similar values for LNFNA, lymph node mononuclear cells (LNMCs) from tissue homogenates by Ficoll-Hypaque separation, and LNFS. Sampling of lymph node viral load by LNFNA appears to capture viral components associated with both individual expressing cells and follicular germinal centers. Due to the advantages in terms of patients' morbidity, repeatability, and cost, assessment of lymphoid tissue viral load by LNFNA warrants an in vivo feasibility trial as an alternative to lymph node biopsy.
Assuntos
DNA Viral/análise , Proteína do Núcleo p24 do HIV/análise , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Linfonodos/virologia , RNA Viral/análise , Adulto , Biópsia por Agulha , Feminino , Produtos do Gene gag/genética , HIV-1/genética , HIV-1/imunologia , Humanos , Hiperplasia , Imuno-Histoquímica , Hibridização In Situ , Linfonodos/química , Linfonodos/patologia , Masculino , Reação em Cadeia da PolimeraseRESUMO
Interleukin-7 (IL-7) has previously been shown to increase antigen-specific immune responses; the effect of IL-7 on human antigen-specific T cell lines has not directly been addressed. A tetanus-toxoid (TT)-specific T cell line exhibited increased proliferation in the presence of exogenous IL-7, suggesting that IL-7 may be useful in the potentiation of immune responses to defined microbial antigens. Murine retroviral vectors encoding the human IL-7 gene and the neomycin phosphotransferase gene (neoR) were packaged into murine retroviral particles, and supernatants containing these retroviral vectors were used to infect a CD4+ lymphoblastoid cell line. Stable integration of the retroviral vector and constitutive expression of the IL-7 gene were observed. Successful IL-7 gene transduction into TT-specific T cells was also accomplished. Detection of neoR DNA sequences and expression of IL-7-specific mRNA increased with selection in geneticin. Production of IL-7 in these cells was induced by exposure to TT. Production of IL-4, IL-6, and interferon-gamma (IFN-gamma) was detected after antigenic stimulation; there was, however, no effect of IL-7 on the pattern or kinetics of cytokine production by these cells. Human IL-7 transduced cells showed greater proliferation to TT than control T cells, particularly at subthreshold TT concentrations. These dta imply that genetic modification of antigen-specific T cells may be a plausible strategy for the study and manipulation of the immune responses to microbial pathogens.