RESUMO
The post-genomics scientific era has evolved rapidly while achieving advanced understanding of the structure and function of the genes responsible for both the phenotypic characteristics of higher organisms and the pathophysiology of several genetic diseases. Researchers in the fields of oncology and infectious diseases have become more convinced of the great potential of molecular biology approaches to further develop highly specific diagnostic and less toxic therapeutic strategies. During the last two decades, approaches for the specific silencing of essential viral genes and cellular oncogenes were evaluated with optimism for developing directed therapies. However, there were drawbacks in the use of antisense oligonucleotides as a practical mechanism of achieving gene silencing both in vitro and in vivo. Recently, a novel role for post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA) was discovered in the experimental model of C. elegans. This mechanism, termed RNA interference (RNAi) has also been found in other eukaryotes, from plants to mammals, including humans. RNAi is presently being explored both in vitro and in vivo in functional genomics studies and possible therapeutic uses due to its highly specific and physiologic mode of gene silencing. This article focuses on the most current information available regarding the RNAi mechanism and its uses in models of cancer and infectious diseases.
Assuntos
Humanos , Animais , Interferência de RNA/fisiologia , Biologia Molecular , Terapia Genética/métodos , Células Eucarióticas/fisiologia , Inativação Gênica/fisiologiaRESUMO
This work reviews the suggested mechanisms which result in programmed cell death in human HIV infection. Here we present state-of-the-art scientific information related to the newly rediscovered phenomenon of Apoptosis, and to its biological relevance in the pathogenesis of HIV disease. General features of this phenomenon are reviewed, as well as available evidence for its occurrence and possible role in AIDS pathogenesis. A complex series of cellular and molecular events leading to cellular apoptosis are also reviewed and discussed. They include events which take place at the cell membrane level and those which occur at the intramembrane level and cytoplasmic locations, which result from the immunological activation of affected cells. Cellular events which follow and occur within the mitochondrial space and at the nuclear level are also discussed. The biological significance of all these phenomena is summarized in a theoretical scheme, which attempts to integrate all cellular events leading a primed cell into its HIV-induced programmed death.
Assuntos
Apoptose , Infecções por HIV/patologia , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/patologia , Antígenos CD4/imunologia , Dano ao DNA , Infecções por HIV/genética , Infecções por HIV/imunologia , Humanos , Ativação Linfocitária , Mitocôndrias/metabolismo , Óxido Nítrico/metabolismo , PesquisaRESUMO
A tri-functional in vitro evaluation has been utilized to analyze peripheral blood mononuclear cells (BMNC) from HIV-infected patients, which allows for the classification of these individuals into convenient stages, according to the number of in vitro parameters affected. The classifying functional parameters are: the mitochondrial metabolic activity of freshly isolated BMNC, measured by an MTT reduction assay, the detection of apoptosis in 72 hour cultures of these cells assessed by propidium iodide staining and dual parametric flow cytometric analysis, and their proliferative response to pokeweed mitogen. Our results indicate that HIV-infected patients at different stages of their clinical disease, can present dysfunctions in one, two or three of the above-mentioned parameters. Based on these results, patients can be classified into four newly-described stages which are Stage 0, including uninfected controls and all patients with unaffected parameters, and Stages 1, 2 and 3, including patients having one, two or all three parameters affected, respectively. This type of immunological evaluation and classification of HIV-infected patients has the potential of becoming a predictive tool in the longitudinal follow-up of their HIV infection.
Assuntos
Soropositividade para HIV/classificação , Soropositividade para HIV/fisiopatologia , Apoptose , Colorimetria , Feminino , Citometria de Fluxo , Humanos , Testes Imunológicos , Leucócitos Mononucleares/metabolismo , PrognósticoRESUMO
Treatment of Rhesus monkey peripheral blood lymphocytes and IL-2 dependent cell lines with heat prior to incubation with mitogens or IL-2, respectively, induces significant cell changes at the nuclear level, detected by DNA staining with Vindelov's propidium iodide and the simultaneous measurement of its red fluorescence and 90 degrees light scatter. These changes are an increase in their nuclear granularity and in apparently fragmented DNA which shows less fluorescence intensity than DNA from nuclei in the G0G1 phase, a phenomenon suggestive of apoptosis. Treated cells also show an increased number of nuclei in G1 or early S phase, with a reduction in those reaching the G2 or M phases. After heat-shock treatment, CTLL-2 cells show an increase in their response to low doses of recombinant IL-2 and an impaired ability to proliferate at higher IL-2 concentrations. These results provide further evidence for the regulatory role of stress-induced events.
Assuntos
Proteínas de Choque Térmico/farmacologia , Interleucina-2/metabolismo , Linfócitos/efeitos dos fármacos , Animais , Ciclo Celular , Núcleo Celular/efeitos dos fármacos , Concanavalina A/farmacologia , Citometria de Fluxo , Temperatura Alta , Linfócitos/metabolismo , Macaca mulattaRESUMO
Infection of the rhesus monkey (Macaca mulatta) with retroviruses originating from African non human primates (SIV) induces in this species an acquired immunodeficiency syndrome (SAIDS) closely resembling AIDS in humans. Analogies between the SIV-rhesus system and AIDS in humans are described in this work, analyzing the close relationship existing between the HIV and SIV viruses, and the similarities between SIV disease in the rhesus and HIV disease in humans. A review of current advances in AIDS vaccine research, using the SIV-rhesus model, is also included.
Assuntos
Síndrome da Imunodeficiência Adquirida , Síndrome de Imunodeficiência Adquirida dos Símios , Síndrome da Imunodeficiência Adquirida/microbiologia , Animais , Modelos Animais de Doenças , HIV/genética , Humanos , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/microbiologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia , VacinasRESUMO
The physical parameters of nuclear size and granularity, and the biological parameters of nuclear protein and DNA content were simultaneously measured by the flow cytometric analysis of mitogen-activated Rhesus monkey lymphocytes. These experiments were performed with the purpose of establishing baseline values for mitogenic assays of Rhesus monkey blood lymphocytes, when examined by the flow cytometric analysis of their nuclear DNA and protein content, and their nuclear light scatter characteristics. Significant proliferative responses to the mitogens PHA, PWM, Con A, and LPS were observed in these experiments. Simultaneous value determinations of DNA content/size, protein content/granularity, DNA content/granularity and DNA/protein content, showed a direct correlation among them, for all mitogens utilized. These results demonstrate the validity of assessing those parameters in the study of in vitro simian lymphocyte activation and that these assays provide a promising tool for the evaluation of changes in lymphocytic function caused by some viral infections in this experimental model.
Assuntos
Linfócitos B/citologia , Macaca mulatta/sangue , Linfócitos T/citologia , Animais , Ciclo Celular , Núcleo Celular , Citometria de Fluxo , Ativação LinfocitáriaRESUMO
The reactivity of seven monoclonal antibodies against the surface antigens of the murine myeloma cell line Sp2/O-Ag 14 was simultaneously analyzed by fluorescence microscopy and flow cytometry. To 1 X 10(6) Sp2/O-Ag 14 cells were added 200 microliters of the monoclonal antibody and the mixture was incubated at 4 degrees C for 30 min. After washing twice with PBS, the Sp2/O-Ag 14 cells were incubated at 4 degrees C for 30 min with a 1:400 dilution of fluoresceinated goat anti-mouse antibodies. Sp2/O-Ag 14 cells were ready for analysis after washing the cells 3 X with PBS. By fluorescence microscopic analysis different patterns of reactivity with monoclonal antibodies were detected. These patterns were identified as: smooth annular, dot-like annular, dot-like patches, diffuse and homogeneous. The observed patterns may represent different cell surface epitopes being recognized by the monoclonal antibodies. Flow cytometry analysis with the EPICS V system showed reactivity of seven monoclonal antibodies with Sp2/O-Ag 14 cell surface epitopes, which ranged from 79 to 90%. Compared to fluorescence microscopy, flow cytometry provides a faster, more sensitive and more accurate quantitative measurement of the reactivity of different monoclonal antibodies against Sp2/O-Ag 14 cell surface antigens.
Assuntos
Anticorpos Monoclonais/análise , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Citometria de Fluxo , Microscopia de Fluorescência , Animais , Camundongos , Células Tumorais Cultivadas/imunologiaRESUMO
The hybridoma technology developed by Kohler and Milstein allows the obtention of antibodies of a single specificity (i.e. monoclonal antibodies). This methodology was first introduced in the Immunology Laboratory of the Department of Microbiology at the Medical Sciences Campus (MSC), University of Puerto Rico (UPR) in 1986. The production of monoclonal antibodies to Sp2/O-Ag 14 reported herein describes the procedure used and is of importance because it constitutes the first work on monoclonal antibodies performed at our institution and in Puerto Rico.