RNA interference: a novel and physiologic mechanism of gene silencing with great therapeutic potential

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.

Characteristics of nitric oxide-induced apoptosis and its target cells in mitogen-stimulated peripheral blood mononuclear cells from HIV+ subjects.

The phenomenon of apoptosis observed in lymphoid cells from HIV+ subjects is an important factor contributing to their massive depletion. Several studies have identified nitric oxide (NO) as one of the molecules involved in the apoptosis phenomenon observed during HIV infection. It has been shown that HIV-derived gp120 enhances NO synthesis in cultured cells from HIV+ individuals. Therefore, we tested the potential of two nitric oxide synthase (NOS) inhibitors with different mechanisms of action as preventive agents of in vitro apoptosis, in peripheral blood mononuclear cells (PBMC) from HIV+ subjects. PBMC isolated from these patients always showed higher apoptosis levels than normal subjects, a fact that correlated with overproduction of NO and with reduction of mitochondrial transmembrane potential in these cells. We identified the CD8+ T lymphocyte sub-population as the major apoptosis target in PBMC cultures. Treatment with NO inhibitors N(G)-monomethyl-L-arginine (L-NMMA) and dexamethasone (DEX) inhibited spontaneous and mitogen-induced apoptosis, while reducing mitochondrial alterations in PBMC from both normal (30%) and HIV+ (70%) subjects. The development of apoptosis in target cells correlated with their mitochondrial transmembrane potential impairment and with increased expression of Fas (CD95) molecules. These results offer additional alternatives for the manipulation of cellular depletion in HIV disease.

Cyclin levels during TNF-alpha-induced apoptosis in HIV-infected cells.

Cyclins are cell cycle regulatory proteins. We compared the concurrent kinetics of apoptosis and cyclin expression between HIV-infected cells (J1.1), and uninfected Jurkat cells. Cells were cultured with TNF-alpha and harvested at 24, 48 and 72 hr to examine cyclin expression and DNA content. We found a decline in the levels of the mitotic B cyclin in Jurkat cells (16 to 2%, 48 hr), while in J1.1 cells it was observed in cyclin E (60 to 37%, 72 hr). Because cyclin B is mitotic, results suggest that Jurkat cells undergo apoptosis at G2, while J1.1 cells enter mitosis and then die by apoptosis, as no changes in cyclin B or DNA content at G2M were observed. G1 cyclin E decline in J1.1 cells also suggests that they die after entering mitosis. Based on differences in the cyclins involved, it seems that HIV-1 manipulates the cell cycle to protect J1.1 cells from apoptosis induction at G2, a critical cell cycle phase for HIV replication. Thus, cyclins are useful to characterize points in the cell cycle at which apoptosis is induced, and could become excellent tools to evaluate mechanisms of action of antiretroviral drugs in the cell cycle of HIV-infected cells.

Decreased levels of TNF-beta in cultured PBLs from HIV+ patients occur concomitantly to increased apoptosis and impaired PWM-induced proliferation and dehydrogenase activity.

The occurrence of apoptosis in cultured blood cells from HIV+ patients is well-documented. However, the relationship of this process to cytokine production is still undefined. We measured the production of TNF-beta by mitogen-stimulated PBLs from 33 HIV+ patients, while simultaneously assessing their development of apoptosis, dehydrogenase activity and proliferative responses to PWM and PHA; Only 3/33 patients had less than 30% apoptosis in PWM cultures (average value = 19.6%); patients were grouped in accordance with their having low (31-40%; average 34.8%), intermediate (41-50%, average 45.7%) or high levels (over 51%, average 53.6%) of apoptosis. Our results indicate that there is a quantitative correlation between the different degrees of apoptosis and the impaired production of TNF-beta, and support the hypothesis that PBLs from all HIV+ patients have defective immunological functions. In addition, our results show that TNF-beta production correlates with a stage classification of patients which reflects their PBL status of apoptosis, proliferative response to PWM and dehydrogenase activity in vitro.

Why do cells die in HIV infection? Potential mechanisms inducing programmed cell death/apoptosis.

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.

Multifunctional immunological monitoring of HIV positive patients: a novel staging system.

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.

Effect of heat-shock on rhesus monkey lymphocytes and IL-2-dependent cells.

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.

[Brief history of AIDS in non-human primates and its contribution to the study of acquired immunodeficiency syndrome]. / Breve historia del SIDA en los primates no humanos y su contribución al estudio del síndrome de inmunodeficiencia adquirida.

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.

The combined assessment of cellular apoptosis, mitochondrial function and proliferative response to pokeweed mitogen has prognostic value in SIV infection.

Using the assessment of the mitochondrial metabolic activity of freshly isolated blood mononuclear cells, the flow cytometric detection of apoptosis and of the proliferative responses to PWM, SIV-infected macaques were classified in: stage 0, which included all animals with unaffected parameters, and stages 1, 2, and 3, which included animals having one, two, or all three parameters affected, respectively. This novel three-parametric staging system (ISS) provides a new prognostic tool in the longitudinal study of SIV infection.

Multiparametric analysis of nuclei from activated B and T lymphocytes of rhesus monkeys.

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.

Assessment of cellular activation by flow cytometric methods.

The in vitro measurement of lymphoid cell activation is a valid correlate of immunological function in human subjects. This process can be evaluated by the assessment of the proliferative response or the cytokine synthesis and secretion by lymphoid cells upon their proper stimulation. The technology of flow cytometry provides unique conditions for the development of biological assays to achieve those purposes. In this work we describe the utilization of flow cytometric DNA analysis in the evaluation of: (1) the mitogenic response of lymphoid cells; (2) the cellular activity in mixed lymphocyte cultures; (3) the presence of interleukin-2 in culture supernatants, and (4) the cell cycle progression, simultaneous to the expression of a nuclear activation antigen among proliferating lymphocytes.

Analysis by fluorescence microscopy and flow cytometry of monoclonal antibodies produced against cell surface antigens.

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.

Production of monoclonal antibodies to Sp2/O-Ag 14 myeloma cells.

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.

Effect of splenectomy on Trypanosoma lewisi infection in young rats.

The effect of splenectomy on animals infected with Trypanosoma lewisi is unclear, and previous reports are inconclusive or conflictive. We splenectomized rats of different ages after they had been infected with T. lewisi. Female Sprague-Dawley rats, Hemobartonella-free, were assigned to four groups according to weight: 80, 108, 140, and 170 g. Each group had splenectomized, sham-operated, and nonoperated control subgroups, all infected with T. lewisi (0.5 ml of 10(6) parasites per ml) 90 h before surgery. Before surgery, parasite levels in host blood were similar. At 24 h after splenectomy in all groups, regardless of weight, blood parasite levels were much higher than they were in sham-operated or control animals (P less than 0.001 to P less than 0.0001; analysis of variance). Younger rats (80 and 108 g) had a higher mortality rate after splenectomy than sham-operated and control animals. Older rats (150 and 170 g) had no mortality. These results show the impact of age and the importance of the spleen on parasite-host interactions in rats infected with T. lewisi.

Lack of greater seroconversion of rhesus monkeys after subcutaneous inoculation of dengue type 2 live-virus vaccine combined with infection-enhancing antibodies.

Four groups of six nonimmune male rhesus monkeys were inoculated subcutaneously with formulations of dengue type 2 vaccine virus DEN-2/S-1. Group A received 1.9 x 10(4) plaque-forming units of vaccine in normal human serum albumin diluent. Group B received the same dose combined with a dengue type 2-immune human serum diluted 1:1,600, beyond its neutralization endpoint of 1:300, but having an immune enhancement titer of 250,000. Groups C and D received 10-fold dilutions of these respective formulations. No migration-inhibitory factor was found when peripheral blood mononuclear leukocytes obtained on day 68 post-immunization from monkeys of all experimental groups were tested. No viremia was detected in any of the monkeys when sera taken on postvaccination days 1 through 12 were inoculated into adult Toxorhynchites amboinensis mosquitoes and LLC-MK2 cells. By day 89, four of the six monkeys had seroconverted by the neutralization test in each of groups A, B, and C, and three of five monkeys in group D (one monkey died from cardiac collapse after anesthesia) had seroconverted. Immune enhancement of dengue virus infection is known to occur in humans and monkeys circulating heterologous flavivirus antibodies. In this study, there was no enhancing effect when antibody was mixed with dengue type 2 vaccine virus and injected subcutaneously.

Leukocyte migration inhibition in vitro in bladder carcinoma.

The leukocyte migration inhibition response in vitro of peripheral blood leukocytes from patients with transitional cell carcinoma of the urinary bladder, other genitourinary diseases, and normal controls was studied. The agarose droplet migration inhibition method was used to determine the cell-mediated immune reactivity of bladder tumor patients to antigens derived from allogeneic bladder tumor cell lines by hypertonic potassium chloride extraction. Sixty-nine bladder tumor patients were tested 131 times and included 68 positive and 63 negative results. A positive assay was shown to correlate strongly with the presence of tumor at the time of testing (p less than 0.001). Five of 24 patients with other genitourinary diseases had positive inhibition assays, but none of the 26 normal controls were positive. These results suggest that this in vitro assay method may prove valuable in monitoring bladder tumor patients for completeness of initial tumor resection and for clinical recurrence as well as for further studies of the tumor-specific immune response in this disease.