Browsing by Author "Rahbar, Elaheh"

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    Allele-specific methylation in the FADS genomic region in DNA from human saliva, CD4+ cells, and total leukocytes
    Rahbar, Elaheh; Waits, Charlotte M. K.; Kirby, Edward H.; Miller, Leslie R.; Ainsworth, Hannah C.; Cui, Tao; Sergeant, Susan; Howard, Timothy D.; Langefeld, Carl D.; Chilton, Floyd H. (2018-04-06)
    Background Genetic variants within the fatty acid desaturase (FADS) gene cluster (human Chr11) are important regulators of long-chain (LC) polyunsaturated fatty acid (PUFA) biosynthesis in the liver and consequently have been associated with circulating LC-PUFA levels. More recently, epigenetic modifications such as DNA methylation, particularly within the FADS cluster, have been shown to affect LC-PUFA levels. Our lab previously demonstrated strong associations of allele-specific methylation (ASM) between a single nucleotide polymorphism (SNP) rs174537 and CpG sites across the FADS region in human liver tissues. Given that epigenetic signatures are tissue-specific, we aimed to evaluate the methylation status and ASM associations between rs174537 and DNA methylation obtained from human saliva, CD4+ cells and total leukocytes derived from whole blood. The goals were to (1) determine if DNA methylation from these peripheral samples would display similar ASM trends as previously observed in human liver tissues and (2) evaluate the associations between DNA methylation and circulating LC-PUFAs. Results DNA methylation at six CpG sites spanning FADS1 and FADS2 promoter regions and a putative FADS enhancer region were determined in two Caucasian cohorts of healthy volunteers: leukocytes in cohort 1 (n = 89, median age = 43, 35% male) and saliva and CD4+ cells in cohort 2 (n = 32, median age = 41, 41% male). Significant ASM between rs174537 and DNA methylation at three CpG sites located in the FADS2 promoter region (i.e., chr11:61594865, chr11:61594876, chr11:61594907) and one CpG site in the putative enhancer region (chr11:61587979) were observed with leukocytes. In CD4+ cells, significant ASM was observed at CpG sites chr11:61594876 and chr11:61584894. Genotype at rs174537 was significantly associated with DNA methylation from leukocytes. Similar trends were observed with CD4+ cells, but not with saliva. DNA methylation from leukocytes and CD4+ cells also significantly correlated with circulating omega-6 LC-PUFAs. Conclusions We observed significant ASM between rs174537 and DNA methylation at key regulatory regions in the FADS region from leukocyte and CD4+ cells. DNA methylation from leukocytes also correlated with circulating omega-6 LC-PUFAs. These results support the use of peripheral whole blood samples, with leukocytes showing the most promise for future nutrigenomic studies evaluating epigenetic modifications affecting LC-PUFA biosynthesis in humans.
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    Engineered models of the lymphatic stroma to study cell and fluid transport
    Hammel, Jennifer H. (Virginia Tech, 2024-11-18)
    The lymphatic system plays essential roles in regulating fluid balance and immunosurveillance. Across the body, local lymphatic vessels collect waste in the form of lymph and deliver it to nearby lymph nodes (LNs) to be filtered and screened for pathogens. With broad implications in adaptive immunity, cancer metastasis, and cancer treatment, developing novel in vitro models will provide new platforms to explore lymphatic function in health and disease. This dissertation sought to develop tissue-specific engineered models of the LN stroma and the meningeal lymphatics to examine the transport of cells and fluid. Within the LN, fibroblastic reticular cells (FRCs) maintain a network of extracellular matrix conduits that guide varying rates of interstitial fluid flow (IFF) based on inflammatory state. Eventually, that flow exits the LN through the afferent lymphatics, consisting of lymphatic endothelial cells (LECs). We first developed a spatially organized model of the LN stroma consisting of a monolayer of LECs on the underside of a tissue culture insert and an FRC-laden hydrogel within. We demonstrate that high magnitude IFF (3.0 µm/s) had positive impacts on FRCs but disrupted the integrity of the LEC barrier, and these effects were accompanied by increased secretion of a variety of inflammatory chemokines. We also show that IFF of any magnitude decreased T cell egress from the model. Next, we sought to apply the LN stroma model toward understanding metastasis. LN metastasis is the most important prognostic factor in breast cancer, with size of metastasis informing treatment plan. Metastasis greatly alters the structure of the LN, which in turn alters transport. However, the impact of altered transport on cancer progression is not well understood. We added different numbers of breast cancer cells to our LN stroma model to examine tumor burden. We found that tumor cells invaded the LEC barrier at similar numbers regardless of initial burden. Additionally, at the highest tumor burden, diffusivity in the stroma was significantly decreased. Most excitingly, flow velocity was positively correlated with FRC spread in the hydrogel, demonstrating the contributions of FRCs to transport. Finally, we looked to the central nervous system (CNS). The meningeal lymphatics are responsible for draining cerebrospinal fluid to the cervical lymph nodes for CNS immunosurveillance. We developed a simple model of a meningeal lymphatic vessel lumen consisting of a monolayer of LECs on the underside of a tissue culture insert and a monolayer of meningeal fibroblasts within. This is, to our knowledge, the very first in vitro model of the meningeal lymphatics. We demonstrate that our model has barrier function and is capable of immune cell transmigration and egress. We examined how systemic chemotherapy for breast cancer could cause off-target disruption of the meningeal lymphatics and found that docetaxel was significantly deleterious. We further began to explore leukemia cell behavior in our LN stroma and meningeal lymphatics model. Throughout this dissertation, we emphasize the importance of incorporating fluid and cell transport into engineered models of immunity. These models represent a step toward building up the complexity of in vitro lymphatic models to improve pre-clinical screening and understand pathophysiology.
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    Label-free analysis of physiological hyaluronan size distribution with a solid-state nanopore sensor
    Rivas, Felipe; Zahid, Osama K.; Reesink, Heidi L.; Peal, Bridgette T.; Nixon, Alan J.; DeAngelis, Paul L.; Skardal, Aleksander; Rahbar, Elaheh; Hall, Adam R. (Springer Nature, 2018-03-12)
    Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the current state of the art.