I would like to thank Sally McDonnell Barksdale Honors College for giving me this incredible opportunity to engage in research and produce this thesis. I would like to thank the Sally McDonnell Barksdale Honors College again for providing the funds to purchase the materials needed for this project. Most importantly, I would like to thank Dr. Paris, my research advisor, for believing in my aspirations and goals;.
Finally, I would like to thank my mother, Artis Young, who is and always will be my greatest. The incidence of HIV among men has been in a stable inclination during the last decades, yet the accelerated rate of infection among older women is significant. One of its viral proteins, Tat, acts as an excitotoxin and disrupts the cell's membrane potential by increasing the concentration of Ca2+ inside the cell.
This influx of calcium ions leads to apoptosis and secretion of the cell's contents into the surrounding area. Restoration of estrogens post-menopausally may improve the incidence of HIV-related neurocognitive impairments that occur with age.
INTRODUCTION
Specifically, infection rates in young women are eight times higher than those in young men (Harrison et al., 2015). In the central nervous system, the virus mainly activates innate immune responders that produce cytokines (Nasi et al., 2014). The BBB provides biological, physiological, and immunological separation between the peripheral system and the central nervous system (Strazza et al., 2011).
However, within the same year, it was discovered by another research committee that neuroAIDS could also be found within the HIV community (Carvour et al., 2015). In fact, nearly half of HIV-infected individuals who had not progressed to AIDS were diagnosed with neurological complications due to NeuroAIDS (Carvour et al. 2015; Saylor et al., 2016). One of the events measured was the time between HIV and AIDS diagnoses; females were observed to have less time than males (Carvour et al., 2015).
Finally, the overall survival time from initial diagnosis to death was lower than men (Carvour et al., 2015). Estrogen is most commonly known as a steroid hormone involved in sexual differentiation and development of males and females (Wilson et al., 2006). ER-beta expression is distributed throughout the CNS, but predominantly in the hippocampus, a brain region largely associated with emotion and cognition (Wilson et al., 2006).
A study conducted in Europe indicated that women were twice as likely to develop neuroAIDS as men (Chiesi et al., 1996). The HIV-1 Tat protein is thought to play an important role in HIV neuropathology, and hormone levels in women may influence these effects (Adams et al., 2010). An experiment conducted using neural cells determined that estrogen was able to delay cell death by narrowing the apoptotic signal produced by Tat (Adams et al., 2010).
Using specific antagonists, it was discovered that ER-beta signaling was preferred, but ER-alpha signaling still contributed to the neuroprotection (Adams et al., 2010). The team investigated the effects of ER-beta, but the resulting levels were almost negligible compared to the resulting levels of ER-alpha (Heron et al., 2009). Due to estrogen's considerable anti-inflammatory and neuroprotective properties, it should be considered as a therapeutic method to counteract inflammation in the brain of HIV-infected menopausal women (Dye et al., 2012).
As there are varying reports and observations on the benefits of estrogen on the central nervous system, it has been hypothesized that estrogen levels are maintained throughout and after menopause in older women (Dye et al., 2012). One study found that a group of older HIV-infected women receiving hormone replacement therapy had a significantly reduced risk of mortality (Dye et al., 2012).
MATERIALS AND METHODS
- DESCRIPTION OF CELLS AND CELL CULTURE
- CHEMICALS INVOLVED IN EXPERIMENTS
- EXPERIMENTAL PROCEDURES USED ON CELLS….14
- ROS ASSAY
- LIVE/DEAD ASSAY
- CYTOKINE/CHEMOKINE ARRAY
Before the cells were collected, several 24-well plates were made with a specific chemical mixture called Poly-L-Lysine. The final wash was removed and then allowed to air dry in the hood overnight. The mixed glial cells were collected from newborn to 2-day-old C57 mouse pups and placed in petri dishes.
When the cells in the 24- or 96-well plates grew at a steady rate, they were required to differentiate into neuronal cells. This was a clear sign that the cells were ready to be treated with the chemicals for the experiment. Cells were intubated with drugs for 20 h and then fluorescence was assessed in the presence of 5-(yn-6)-chloromethyl-2'7'-.
Briefly, cells were loaded with 10 µM CM-H2DCFDA in warm HBSS for 45 min (according to manufacturer's protocol), and washed twice. The goal of the cytokine protein array was to screen potentially upregulated cytokines and chemokines to discover future analytical targets. Proteim Profiler Mouse Cytokine Array Kit, #ARY006, R&D Systems, Minneapolis, MN) assessed the protein content of the following analytes.
Given that phenol red (a common component included in cell culture media) contains phytoestrogenic activity, cells were evaluated after incubation in media containing phenol red or media without phenol red. Cells grown in phenol red and treated with additional estrogen agonists showed less ROS production than cells in media without phenol red. The inclusion of estrogen receptor agonists along with the natural estrogen supplements of phenol red medium appeared to create additional protection against the neurotoxic effects of Tat and significantly reduced cell death.
Since the phenol red-free medium does not contain any estrogen supplements, even a small addition of estrogen receptor agonists was not sufficient to provide sufficient protection, resulting in increased ROS production and cell death. There was a significant interaction [F p<0.05] such that Tat significantly increased cell death when grown in medium containing phenol red (Figures 2a and b). The purpose of testing Tat under these conditions was to determine the indirect neurotoxicity of the viral protein.
DISCUSSION
The media of the ROS assay was then manipulated to further support the hypothesis that estradiol would attenuate these effects. One study pursued this question and determined that ER-beta was the primary source of protection (Adams et al., 2010). It was discovered that ER-beta signaling was preferred, but ER-alpha signaling still contributed to the neuroprotection (Adams et al., 2010).
Using both enantiomers of the chemical, the team was able to confirm that equol provided neuroprotection in an estrogen-dependent manner and that both acted in an ER-beta-dependent mechanism (Bertrand et al., 2015). Rather, neuronal damage must occur by indirect mechanisms, including those caused by toxic viral proteins, such as Tat (King et al., 2006). The secretion of Tat from infected cells into local tissues activates surrounding microglial cells, leading to the secretion of defense chemicals: cytokines and chemokines (King et al., 2006).
Tat induced some anti-inflammatory cytokines, but most of the stimulated cytokines were anti-inflammatory. CCL2 is a chemokine that stimulates the immune system to sites of tissue damage or infection (Deshmane et al., 2009). Finally, they observed a significant increase in the chemokine MCSF, which happens to be related to the aforementioned CCL2.
The protocol used in this experiment was not maintained throughout the duration of the study. A portion of the plates had fluorescent dye incorporated into the medium during all time intervals. The second part of the panels was then performed using the appropriate protocol and noted that the death rate decreased.
Future experiments could use the latter protocol, called endpoint analysis, when the fluorescent dye is added at the beginning of the 24-hour time course. Through dose-specific incorporations of estrogen, the entire CNS can be partially protected from the damaging effects of Tat on the nervous tissue of the brain. Roles and functions of HIV-1 Tat protein in the CNS: an overview" Virology journal vol.
Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research vol. Zhang, YL, et al., “Blood-brain barrier and neuro-AIDS.” European Review of Medical and Pharmacological Sciences, vol.
