Screening assay for oxidative stress in a feline astrocyte cell line, G355-5.

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H(2)DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H(2)DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H(2)DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H(2)DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H(2)DCFDA fluorescence was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.

Methamphetamine and lentivirus interactions: reciprocal enhancement of central nervous system disease.

Use of methamphetamine is increasingly a significant factor for the spread of human immunodeficiency virus type 1, for in certain populations, there is a convergence of methamphetamine abuse with human immunodeficiency virus type 1 infection. Methamphetamine and human immunodeficiency virus type 1 are both individually neuropathogenic, and the neuropathology caused by these two agents occurs in overlapping brain regions. However, the biological interaction of methamphetamine with lentiviruses remains unknown. Here, we investigate the effects of simultaneous exposure of these two agents on disease progression using the feline immunodeficiency virus model. The study models the bingeing methamphetamine user with sequential and repeated episodes of use, which were interrupted by periods of abstinence. Methamphetamine exposure significantly accelerated and enhanced the severity of the feline immunodeficiency virus model-induced central nervous system functional pathology, as measured in delays in brainstem auditory evoked potentials. Reciprocally, feline immunodeficiency virus enhanced the severity of the methamphetamine-induced effects on brain monoamine neurotransmitter and dopamine transporter levels. The results of this study indicate that a dual potentiation occurred. That is, methamphetamine enhanced feline immunodeficiency virus model-induced central nervous system disease and feline immunodeficiency virus model enhanced the toxic effects of methamphetamine, heralding a significant concern for those individuals that are exposed to both agents.

Domain- and nucleotide-specific Rev response element regulation of feline immunodeficiency virus production.

Computational analysis of feline immunodeficiency virus (FIV) RNA sequences indicated that common FIV strains contain a rev response element (RRE) defined by a long unbranched hairpin with 6 stem-loop sub-domains, termed stem-loop A (SLA). To examine the role of the RNA secondary structure of the RRE, mutational analyses were performed in both an infectious FIV molecular clone and a FIV CAT-RRE reporter system. These studies disclosed that the stems within SLA (SA1, 2, 3, 4, and 5) of the RRE were critical but SA6 was not essential for FIV replication and CAT expression. These studies also revealed that the secondary structure rather than an antisense protein (ASP) mediates virus expression and replication in vitro. In addition, a single synonymous mutation within the FIV-RRE, SA3/45, reduced viral reverse transcriptase activity and p24 expression after transfection but in addition also showed a marked reduction in viral expression and production following infection.

Escalating morphine exposures followed by withdrawal in feline immunodeficiency virus-infected cats: a model for HIV infection in chronic opiate abusers.

Opiate abuse is a risk factor for human immunodeficiency virus (HIV) infection. Because the direct effects of opiates on HIV infection are difficult to determine epidemiologically, animal models of lentivirus infection are relied upon to study the effects of opiates in the absence of confounding factors. Morphine, the predominant metabolite of heroin, is used in most experimental systems examining heroin abuse. In this study, morphine treatment of feline immunodeficiency virus (FIV)-infected cats modeled a typical pattern of escalating drug use interspersed with withdrawals. Plasma cortisol levels were measured for evidence of stress associated with morphine withdrawal. In the morphine-treated cats, cortisol levels peaked at time points corresponding to morphine withdrawal and returned to baseline levels during treatment and several weeks after the final withdrawal. Morphine-treated cats displayed clear behavioral and physical signs of opiate exposure and evidence of withdrawal when the drug was stopped. Morphine-exposed cats did not experience enhanced severity of FIV-related disease; in fact, morphine demonstrated a protective effect on FIV-associated changes in brainstem auditory evoked potentials. Our research suggests that opiate exposure is unlikely to adversely affect the progression of acute lentivirus infection and might be beneficial in controlling associated neurological disease.

Effects of 1,8-diaminooctane on the FIV Rev regulatory system.

Proper function of the Rev regulatory system is essential for the replication of lentiviruses, including feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1). Specifically, Rev affects the overall stability of viral mRNAs that encode necessary structural and enzymatic proteins. In turn, the eukaryotic initiation factor (eIF-5A) is indispensable for Rev function and is the only known protein whose biologically active form requires the unique amino acid, hypusine. Because 1,8-diaminooctane blocks the formation of hypusine by disrupting the cellular enzyme, deoxyhypusine synthase, thereby preventing activation of eIF-5A, we investigated the effects of 1,8-diaminooctane on posttranscriptional regulation. These are the first results to demonstrate that diaminooctane significantly reduced viral replication in a dose-dependent manner, even under conditions of contact inhibition, diminishing the compound's effect on cell proliferation. Similarly, the addition of increased concentrations of diaminooctane caused a reduction in the expression of a Rev-dependent CAT system without affecting a Rev-independent CAT system. At the RNA level, exposure of chronically infected CrFK cells to increasing concentrations of diaminooctane substantially decreased the levels of unspliced and singly spliced viral mRNAs and increased the relative amounts of multiply spliced transcripts in the cytoplasm. The findings of this study are the first demonstration that FIV, similar to HIV-1, requires eIF-5A for efficient Rev function and that small molecule intervention can indirectly target this lentivirus regulatory system.

Inhibition of feline immunodeficiency virus (FIV) replication by DNA binding polyamides.

Two DNA minor-groove binding polyamides 1 and 2 were designed and synthesized and evaluated for inhibition of FIV-34TF10 replication. Both 1 and 2 decreased the replication of FIV-34TF10 by 75% by acting at the level of the virus but outside of the LTR or env region.

A malaria vaccine candidate based on a hepatitis B virus core platform.

OBJECTIVE: The recent success of a Plasmodium falciparum malaria vaccine consisting of circumsporozoite (CS) protein (CSP) T and B cell epitopes has rekindled interest in the development of a pre-erythrocytic vaccine. Our goal was to design an efficient delivery system for known neutralizing epitopes. METHODS: Well-characterized CSP-specific neutralizing B cell epitopes and a 'universal' T cell epitope were combined with a particulate carrier platform, the hepatitis B core antigen (HBcAg), to produce a novel pre-erythrocytic vaccine candidate. RESULTS: The vaccine candidate V12.PF3.1 is a potent immunogen in mice, eliciting unprecedented levels (greater than 106 titers) of sporozoite-binding antibodies after only two doses. The antisporozoite antibodies are long-lasting and represent all IgG isotypes, and antibody production is not genetically restricted. CSP-specific CD4+ T cells are also primed by V12.PF3.1 immunization in a majority of murine strains. Furthermore, the hybrid HBcAg-CS particles can be produced inexpensively in bacterial expression systems. CONCLUSION: These characteristics suggest that V12.PF3.1 represents an efficient and economical P. falciparum vaccine candidate for use separately or in combination with other formulations.