Liver fibrosis as a result of Hepatitis C viral infection is characterized by an accumulation of extracellular matrix (ECM) during liver injury. Under chronic stress, fibrosis can develop into cirrhosis with severe levels of ECM based scar tissue and impede normal functions of the liver resulting in its failure. Hepatic stellate cells (HSC) make up approximately 5-8% of the liver’s total composition and, when activated as a response to liver damage, are the main ECM producing cells. Normally, HSC are isolated and purified from an animal liver such as a rat or mouse via an enzymatic digestion of the liver tissue and differential centrifugation. The ability of primary HSC to grow in culture is limited and so the generation of HSC lines has been an important development in examining the biology of HSC . However, most studies investigating the activation of a hepatic stellate cell from their quiescent to myofibroblast-like, ECM-producing, state (characterized by an increasing expression of smooth muscle alpha actin (alpha-SMA)) use the protocol of primary HSC isolation and purification because of the fact that primary HSC undergo spontaneous activation on uncoated plastic. This in vitro activation is thought to be similar to activation in vivo. LX-2, a human HSC line, has been characterized by several studies as at least “partially activated” due to its innate expression of alpha-SMA and therefore raises questions of its effectiveness as model for actual HSC activation. In this study we investigated the use of LX-2 as a means of studying the evolution of HSC activation by seeking a further increase in activation (characterized by alpha-SMA) initiated by hypoxic stress.  Western blot results indicated a slight increase in alpha-SMA expression.  The effect of serum conditions on activation were also tested, but not pursued further due to the low viability of cells in serum-free media.  Based on these results it was concluded that our LX-2 cells were already "too activated" to serve as a sufficient model to study the transition of HSC from a quiescent to activated state.

Prolongation of the QT interval of the heart’s electrical cycle is a potential adverse effect carried by a wide variety of drugs prescribed for various conditions. This effect has been linked to increased susceptibility of torsades de pointes (TdP) and fatal arrhythmias. The Totah lab hypothesizes that one factor may involve an enzyme known as Cytochrome P450 2J2 (CYP2J2), known to be present in the heart and responsible for metabolizing endogenous arachidonic acid into epoxyeicosatrienoic acid (EET) isomers. EETs are physiologically important, especially in the heart, because of their role in providing several protective properties such as vasodilation. Consequently, interference of this enzyme’s activity through xenobiotic inhibition could contribute to QT-prolongation and potentially fatal cardiotoxicity. Here, the activity and inhibition of CYP2J2 are being investigated in four specific models: recombinant enzyme, transgenic mouse hearts, human heart tissue, and human cardiomyocytes through the use of various assays and mass spectroscopy. CYP2J2 expression in each of the models is also being quantified through immunoblotting. Drugs such as pimozide (an anti-psychotic) and danazol (a modified testosterone), which are associated with QT-prolongation, have experimentally inhibited CYP2J2 activity by as much as 50% and 80%, respectively. The results of this research are expected to increase the understanding of CYP2J2’s role in the heart, and the potential role of this isozyme in QT-prolongation and other fatal heart conditions..

The overall goal of the project is to apply high-throughput genomic and virological studies of our novel cohort of seronegative HIV-1 infection controllers (SHIC) to comprehensively define viral, host factors and mechanism(s) associated with disease control. HIV-1 elite controllers (EC) account for approximately 0.3% of HIV-1 infected individuals. EC/SHIC maintain durable control of HIV-1 infection, measured by viral loads below the limits of detection by commercial assays (<50 HIV-1 RNA copies/ml blood plasma). Therefore, this population represents the most relevant population to study control mechanisms of HIV-1 infection. Unfortunately, there is a gap in our understanding on how/if HIV-1 is controlled at early stage(s) because all EC studies to date were performed on patients identified 10 or more years post-infection. To address this gap in knowledge, we have developed technologies that allow for the identification of SHIC/EC near the time of HIV acquisition. We have established a Seattle HIC cohort (n=22) and a SHIC China cohort (n=80) with samples available pre- and post-HIV-1 infection. To identify genes associated with infection control we will expression profile PBMCs from SHIC pre- and post-infection, and compare them to PBMC expression profiles from acute HIV-1 infected individuals (AHI) and uninfected controls. By creating a statistical correlation matrix we will identify differentially expressed genes predictive of infection control. When we compare PBMC profile of SHIC pre-infected to PBMC post-infection, we expect minimal differences, because SHIC have extremely low viral load. Comparing PBMC profiles of healthy controls to AHI will identify a set of virus specific gene patterns in response to infection. We will compare post-infection SHIC PBMC profiles to AHI to yield expression patterns common to SHIC and AHI; subtracting these elements from the preceding set will identify genes overrepresented in SHIC associated with HIV-1 control following acute infection.

This project investigates the importance of type IV pilus (TFP) adhesins of the symbiont Verminephrobacter eiseniae during colonization of the earthworm Eisenia fetida. Earthworms contain symbiotic bacteria within their gut and kidney-like organs known as nephridia. Verminephrobacter eiseniae has adapted to live specifically in worm excretory organs. We hypothesize that while the earthworm provides a thriving environment for the bacteria, the bacteria detoxify wastes and play a role in nitrogen conservation. The bacterial symbionts are vertically transferred by deposition into the host earthworm’s egg capsules, which then colonize the embryonic worm. V. eiseniae, has genes encoding for several modes of movement including chemotaxis, type IV pili (TFP), and flagellar motility. The TFP binds and creates a pulling force by retraction of a hook-like apparatus allowing for bacteria to move over a wide variety of surfaces. We hypothesize that the binding of TFP to host cell surfaces is mediated by adhesins on the pilus tip. V. eiseniae has seven pilus adhesins that may play an important role in binding specifically at distinct phases of the earthworm life cycle. To determine how adhesins mediate symbiotic bacterial binding to specific tissues in the host earthworm, I created gene knockouts by disrupting certain TFP gene components, including but not limited to the tip adhesins. Studying the role of TFP and associated adhesins in the binding process not only provides a greater understanding of the beneficial bacteria-host cell interactions, but also pathogen binding. Pathogenic bacteria also use TFP with specific adhesins to bind host cells in order to initiate the infection process. Investigating the specificity of these bacterial features provides further insight regarding pathogen-host binding. Therefore, our findings may also be applied to treating human pathogens which utilize similar modes of infection.