Brenda Morsey

Mechanism of alcohol-induced oxidative stress and neuronal injury

Neuro-cognitive deficits, neuronal injury, and neurodegeneration are well documented in alcoholics, yet the underlying mechanisms remain elusive. Oxidative damage of mitochondria and cellular proteins intertwines with the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. Here, we present the evidence that metabolism of ethanol in primary human neurons by alcohol dehydrogenase (ADH) or cytochrome P450-2E1 (CYP2E1) generates reactive oxygen species (ROS) and nitric oxide (NO) via induction of NADPH/xanthine oxidase (NOX/XOX) and nitric oxide synthase (NOS) in human neurons. The acetaldehyde-mediated increase in NOX, XOX, or NOS activity is regulated as a transcriptional rather than a translational process. Marked increase in the lipid peroxidation product (4-hydroxynonenal) and enhanced ROS generation coincides with decreased neuronal viability and diminished expression of neuronal marker (neurofilaments). Novel quantitative methods of ROS and NO detection help dissect the mechanisms of alcohol-induced neurodegeneration. Uncovering the basic mechanisms of oxidative neuronal injury will serve as the basis for development of new therapies.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) suppresses Rho GTPases in human brain microvascular endothelial cells and inhibits adhesion and transendothelial migration of HIV-1 infected monocytes.

Under inflammatory conditions (including HIV-1 encephalitis and multiple sclerosis), activated brain endothelium enhances the adhesion and transmigration of monocytes across the blood-brain barrier (BBB). Synthetic ligands that activate the peroxisome proliferator-activated receptors (PPARs) have anti-inflammatory properties, and PPAR stimulation prevents the interaction of leukocytes with cytokine stimulated-endothelium. However, the mechanism underlying these effects of PPAR ligands and their ability to intervene with leukocyte adhesion and migration across brain endothelial cells has yet to be explored. For the first time, using primary human brain endothelial cells (BMVEC), we demonstrated that monocyte adhesion and transendothelial migration across inflamed endothelium were markedly reduced by PPARγ activation. In contrast to non-brain-derived endothelial cells, PPARα activation in the BMVEC had no significant effect on monocyte-endothelial interaction. Previously, our work indicated a critical role of Rho GTPases (like RhoA) in BMVEC to control migration of HIV-1 infected monocytes across BBB. In this study, we show that in the BMVEC PPARγ stimulation prevented activation of two GTPases, Rac1 and RhoA, which correlated with decreased monocyte adhesion to and migration across brain endothelium. Relevant to HIV-1 neuropathogenesis, enhanced adhesion and migration of HIV-1 infected monocytes across the BBB were significantly reduced when BMVEC were treated with PPARγ agonist. These findings indicate that Rac1 and RhoA inhibition by PPARγ agonists could be a new approach for treatment of neuroinflammation by preventing monocyte migration across the BBB.

Chemical and biological pollution contribute to the immunological profiles of free‐ranging harbor seals

Polychlorinated biphenyls and other persistent organic pollutants have been associated with immunotoxicity and outbreaks of (infectious) disease in marine mammals by rendering them vulnerable to infection by pathogens such as viruses and bacteria. In an immunotoxicological study of free-ranging harbor seals (Phoca vitulina), we obtained samples of blood and blubber from seal pups that were live-captured from two remote and two near-urban sites in British Columbia, Canada, and Washington state, USA. Using these samples, we quantified hematology, innate immune function, adaptive immune function, and polychlorinated biphenyl accumulation. While controlling for confounding factors (age, sex, and condition), univariate correlations between phagocytosis (r2 = 0.30, p = 0.002), respiratory burst (r2 =0.45, p= 0.000), T-lymphocyte function (r2 = 0.16, p = 0.028), lymphocyte signaling (r2 = 0.17, p = 0.025), and lymphocyte counts (r2 = 0.29, p = 0.002), and polychlorinated biphenyl concentrations suggested chemical-associated immunotoxicity. Principal component analysis of immunological endpoints provided additional evidence of immunotoxic effects in seals. However, principal component analysis also identified a noncontaminant-related factor by distinguishing between seals inhabiting urban versus remote sites, with results being consistent with increased pathogen exposure. Elevated fecal coliform concentrations in water, and observations of terrestrial spill-over pathogens in local seals, further support the notion of biological pollution at these sites. Although our study highlights the role that environmental contaminants might play in rendering marine mammal populations vulnerable to disease through immunotoxicity, it also suggests that biological pollution represents an emerging conservation concern.

Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins

Mitochondrial dysfunction is implicated in a majority of neurodegenerative disorders and much study of neurodegenerative disease is done on cultured neurons. In traditional tissue culture, the oxygen level that cells experience is dramatically higher (21%) than in vivo conditions (1–11%). These differences can alter experimental results, especially, pertaining to mitochondria and oxidative metabolism. Our results show that primary neurons cultured at physiological oxygen levels found in the brain showed higher polarization, lower rates of ROS production, larger mitochondrial networks, greater cytoplasmic fractions of mitochondria and larger mitochondrial perimeters than those cultured at higher oxygen levels. Although neurons cultured in either physiological oxygen or atmospheric oxygen exhibit significant increases in mitochondrial reactive oxygen species (ROS) production when treated with the human immunodeficiency virus (HIV) virotoxin trans-activator of transcription, mitochondria of neurons cultured at physiological oxygen underwent depolarization with dramatically increased cell death, whereas those cultured at atmospheric oxygen became hyperpolarized with no increase in cell death. Studies with a second HIV virotoxin, negative regulation factor (Nef), revealed that Nef treatment also increased mitochondrial ROS production for both the oxygen conditions, but resulted in mitochondrial depolarization and increased death only in neurons cultured in physiological oxygen. These results indicate a role for oxidative metabolism in a mechanism of neurotoxicity during HIV infection and demonstrate the importance of choosing the correct, physiological, culture oxygen in mitochondrial studies performed in neurons.

Methamphetamine Causes Mitrochondrial Oxidative Damage in Human T Lymphocytes Leading to Functional Impairment

Methamphetamine (METH) abuse is known to be associated with an inordinate rate of infections. Although many studies have described the association of METH exposure and immunosuppression, so far the underlying mechanism still remains elusive. In this study, we present evidence that METH exposure resulted in mitochondrial oxidative damage and caused dysfunction of primary human T cells. METH treatment of T lymphocytes led to a rise in intracellular calcium levels that enhanced the generation of reactive oxygen species. TCR-CD28 linked calcium mobilization and subsequent uptake by mitochondria in METH-treated T cells correlated with an increase in mitochondrion-derived superoxide. Exposure to METH-induced mitochondrial dysfunction in the form of marked decrease in mitochondrial membrane potential, increased mitochondrial mass, enhanced protein nitrosylation and diminished protein levels of complexes I, III, and IV of the electron transport chain. These changes paralleled reduced IL-2 secretion and T cell proliferative responses after TCR-CD28 stimulation indicating impaired T cell function. Furthermore, antioxidants attenuated METH-induced mitochondrial damage by preserving the protein levels of mitochondrial complexes I, III, and IV. Altogether, our data indicate that METH can cause T cell dysfunction via induction of oxidative stress and mitochondrial injury as underlying mechanism of immune impairment secondary to METH abuse.

Immunomodulatory effects of in vitro exposure to organochlorines on T-cell proliferation in marine mammals and mice.

Marine mammals bioaccumulate various environmental contaminants such as organochlorines (OCs), which biomagnify via the food web. While the immunomodulatory effects of individual OCs have been studied, the effects of mixtures are not well understood. The immunomodulatory effects of polychlorinated biphenyl (PCB) 138, 153, 169, and 180 as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and all possible mixtures were examined in marine mammals and mice. Lymphocyte proliferation was significantly modulated by OCs in all species tested, mostly by non-coplanar PCBs, as shown using regression analyses. Correlation analyses showed significant correlations (interpreted as additive effects) between OCs in mice, killer whales, and Steller sea lions. Nonadditive synergistic and antagonistic interactions between OCs were detected in most of the species tested. Toxic equivalency (TEQ) values used for OC toxicity assessment failed to predict the immunomodulatory effects measured in mice and marine mammals. The commonly used mouse model failed to predict immunomodulatory effects in other species. Clustering data suggested that phylogeny does not predict toxicity of OCs. Overall, our data suggest the presence of species-specific sensitivities to different mixtures, in which OCs interactions may be complex and that may exert their effects through dioxinlike or dioxin-independent pathways. Lastly, lymphocyte proliferation, an important part of adaptive immunity, was significantly modulated in mice and marine mammals, suggesting the possibility of increased susceptibility to diseases. These findings will be useful to better characterize the risk associated with OC exposure and possibly lead to new conservation and management strategies.

Non-coplanar PCB-mediated modulation of human leukocyte phagocytosis: a new mechanism for immunotoxicity.

Organochlorine (OC) contaminants, notably polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ubiquitous in all ecosystems and found in the tissues of humans and wildlife. Although the immunotoxicity of coplanar, dioxinlike PCBs is well documented, the adverse effects exerted by non-coplanar, non-dioxinlike PCBs have received little attention. Direct causal relationship between PCB and dioxin exposure and the observed detrimental effects on the immune system has yet to be fully established in humans. The immunomodulatory potential of toxic coplanar PCB 169 and TCDD and abundant non-coplanar PCBs 138, 153, and 180 on human leukocyte phagocytosis, an important innate immune function that initiates the clearance of pathogens, was tested upon in vitro exposure. Mixture and concentration-response experiments demonstrated a suppression of phagocytosis by non-coplanar PCBs suggesting a previously unrecognized aryl hydrocarbon receptor (AhR)-independent pathway. Regression analysis revealed that reduction of phagocytosis was mostly explained by the non-coplanar congeners. The effects on phagocytosis could not be accurately predicted by either the currently used toxic equivalence (TEQ) approach or the mouse model, thus undermining the use of the traditional models in the risk assessment for OC mixtures containing non-coplanar congeners. Our results are cause for concern as they suggest an AhR-independent pathway through which non-coplanar PCBs modulate phagocytosis, the immune systems first line of defense, possibly increasing the risk to developing infectious disease.

Preclinical Pharmacokinetics and Tissue Distribution of Long-Acting Nanoformulated Antiretroviral Therapy

ABSTRACT Long-acting injectable nanoformulated antiretroviral therapy (nanoART) was developed with the explicit goal of improving medicine compliance and for drug targeting of viral tissue reservoirs. Prior nanoART studies completed in humanized virus-infected mice demonstrated sustained antiretroviral responses. However, the pharmacokinetics (PK) and tissue distribution of nanoART were not characterized. To this end, the PK and tissue distribution of nanoformulated atazanavir (ATV) and ritonavir (RTV) injected subcutaneously or intramuscularly in mice and monkeys were evaluated. Fourteen days after injection, ATV and RTV levels were up to 13-, 41-, and 4,500-fold higher than those resulting from native-drug administration in plasma, tissues, and at the site of injection, respectively. At nanoART doses of 10, 50, 100, and 250 mg/kg of body weight, relationships of more- and less-than-proportional increases in plasma and tissue levels with dose increases were demonstrated with ATV and RTV. Multiple-dose regimens showed serum and tissue concentrations up to 270-fold higher than native-drug concentrations throughout 8 weeks of study. Importantly, nanoART was localized in nonlysosomal compartments in tissue macrophages, creating intracellular depot sites. Reflective data were obtained in representative rhesus macaque studies. We conclude that nanoART demonstrates blood and tissue antiretroviral drug levels that are enhanced compared to those of native drugs. The sustained and enhanced PK profile of nanoART is, at least in part, the result of the sustained release of ATV and RTV from tissue macrophases and at the site of injection.

HIV-1 activates proinflammatory and interferon-inducible genes in human brain microvascular endothelial cells: putative mechanisms of blood-brain barrier dysfunction.

The mechanisms underlying blood—brain barrier (BBB) dysfunction seen in human immunodeficiency virus 1 (HIV-1) infection are poorly understood; however, they are believed to be caused by interactions of human brain microvascular endothelial cells (HBMEC) with virus-infected macrophages. Using a transwell system and Affymetrix arrays, we investigated HIV-1-induced genomic changes in HBMEC after coculture with HIV-1-infected or -uninfected monocyte-derived macrophages (MDM). Differentially expressed genes were determined by linear modeling and then were grouped by hierarchical clustering. Compared to HBMEC cocultured with noninfected MDM, 184 probe sets corresponding to 84 genes were differentially expressed in HBMEC cocultured with HIV-infected MDM. Genes activated in HIV-1 MDM-exposed HBMEC included proinflammatory cytokines and chemokines, tumor necrosis factor-α-induced proteins, interferon (IFN)-inducible genes, intercellular adhesion molecule-1, transcription factors of the nuclear factor-κB family, and signal transducer and activator of transcription 1. Analysis of molecular networks and canonical pathways associated with differentially expressed genes suggest that HIV-1 causes BBB impairment by mechanisms involving inflammation, cytokine, and IFN signaling in HBMEC.

IMMUNOMODULATION OF CRASSOSTREA GIGAS AND CRASSOSTREA VIRGINICA CELLULAR DEFENSE MECHANISMS BY PERKINSUS MARINUS

Abstract The eastern oyster is an economically and ecologically important species whose vitality is threatened by the protozoal parasite Perkinsus marinus. To better understand which cellular defense mechanisms impart resistance to P. marinus, resistant (Crassostrea gigas) and susceptible (Crassostrea virginica) oyster species were challenged by an experimental infection with P. marinus and their cellular responses were quantified and compared. Both in vivo and in vitro infection trials measured hemocyte phagocytosis, respiratory burst, apoptosis at 1, 3 and 7 days postinfection (in vivo) or 1-h postco-incubation (in vitro). Total parasite body burden concentrations were also measured at the end of in vivo infections. Infections were significantly more severe in C. virginica than C. gigas at 3 and 7 days postinfection confirming the resistance of C. gigas and validating the experimental model. There was more phagocytosis in infected C. virginica than infected C. gigas three days postinfection. In vitro, C. virginica granulocytes phagocytized significantly more parasites and fluorescent latex beads than C. gigas granulocytes, and infection increased bead phagocytosis in both species, equally in cells with or without intracellular parasites. Neither in vivo nor in vitro infections significantly increased respiratory burst activity. While in vitro infections suppressed hemocyte apoptosis in both species, in vivo infections increased hemocyte apoptosis frequency in C. gigas at 3 days postinfection. In vivo infection increased hemocyte apoptosis in C. virginica at 7 days postinfection but not at three days postinfection. From those experiments, we concluded that the increased phagocytosis without concomitant increase in respiratory burst activity seen in infected C. virginica might exacerbate infections. Also, while in vitro P. marinus infection suppresses hemocyte apoptosis in both species, C. gigas appeared to overcome that suppression faster than C. virginica upon in vivo infection, suggesting that hemocyte apoptosis may be an effective oyster defense response against P. marinus infection. The combination in vitro and in vivo infections in P. marinus disease resistant and susceptible oyster species with multiple time points and assays allowed the identification of apoptosis as the cellular defense mechanism most likely to play an important role in defense against P. marinus. This information may provide more accurate predictive criteria for disease resistance, allowing for the testing and selection of more disease resistant oysters.