Browsing by Subject "Leukocytes, Mononuclear"
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Item Peripheral Blood Gene Expression in Discoid Lupus Erythematosus and Systemic Lupus Erythematosus Patients(2016-01-19) Bishop, Allison; Tseng, Lin-Chiang; Chong, Benjamin F.BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease with variations in clinical presentation that can affect the kidney, blood vessels, heart, lungs, joints, CNS, and/or the skin. The most common cutaneous form is discoid lupus erythematosus (DLE). DLE can occur with or without SLE, and differentiating between the two disease states can be ambiguous. Recent studies have shown type I interferon-related genes to be upregulated in peripheral blood mononuclear cells (PBMCs) of SLE patients and DLE patients compared with normal controls. Other studies have suggested that cytokines could serve as biomarkers of disease progression from DLE to SLE. Identifying differences in the transriptomes of DLE patients versus SLE patients could aid in distinguishing these two groups and forecasting progression from DLE to SLE. AIM: This study aimed to analyze differences in the expression levels of type I interferon-related genes and cytokine genes in whole blood samples of DLE patients, SLE patients, and normal controls. We hypothesized that SLE patients would have increased expression levels of these genes compared to DLE patients, due to their widespread systemic disease. METHODS: Blood samples were collected from DLE, SLE, and normal patients recruited at the outpatient UTSW and Parkland Hospital Dermatology clinics. Reverse transcription polymerase chain reaction (RT-PCR) analysis of ten genes, including five type I interferon-related genes (MX1, LY6E, OAS1, OASL, and ISG15) and five cytokine genes (CXCL10, TNF-α, IL-6, IL-10, and IL-12), was performed. RNA was extracted using RNeasy Lipid Tissue Mini kit, and the RNA was reverse transcribed to cDNA. cDNA of selected genes was amplified with SYBR Green PCR Master Mix using forward and reverse primers. Cycle threshold (Ct) values were standardized to the housekeeping gene GAPDH and converted to fold change using the 2-ΔΔCT formula. Gene expression levels were compared between these groups using the Kruskal-Wallis test. RESULTS AND CONCLUSIONS: All of the interferon-related genes were found to be significantly overexpressed (p<0.05) in the whole blood of DLE and SLE patients compared to normal controls. However none of the interferon-related genes showed a significant difference in gene expression between DLE and SLE patients. Expression of cytokine genes either did not show any significant differences between groups or was too low to detect. Future directions include RT-PCR analyses of cytokine gene expression of stimulated patient PBMCs, and whole blood gene expression of other candidate biomarker genes identified from our whole blood microarray data.Item Toward the Rational Design of Better Antivirals: The Development of cGAMP as an HIV-1 Anti-Retroviral and the Genetic Surveillance of WNV Evolution(2017-04-17) Aroh, Chukwuemika Nnabuike; Pasare, Chandrashekar; Wakeland, Edward K.; Yan, Nan; Schoggins, John W.; Pfeiffer, Julie K.The innate immune response is the first line of defense against pathogens and thus represents the first hurdle viruses must overcome to cause severe disease in humans. Understanding the consequences of viral evolution can give insights to mechanisms of viral pathogenesis as well as the development of novel therapeutics. Here I studied two clinically important viruses: Human Immunodeficiency Virus (HIV) and West Nile Virus (WNV). HIV-1 has evolved several mechanisms to evade immune detection by the cGAS-STING cytosolic DNA sensing pathway. A small cyclic di-nucleotide, cGAMP, activates the same pathway by directly binding STING. Treatment with cGAMP, delivered by ultra-pH sensitive nanoparticles or by liposomes, in human peripheral blood mononuclear cells (PBMCs) induced potent and long-acting protection against replication of several laboratory-adapted and clinical HIV-1 isolates in contrast to the short-lasting effect of current anti-retroviral therapy (ARTs). These results present the first evidence for potentially developing cGAMP or other STING agonists as a long-acting antiretroviral immunotherapy. West Nile Virus (WNV) is a mosquito-borne Flavivirus which was introduced to North America in 1999 and is currently the leading cause of viral encephalitis. The lack of specific therapeutics or human vaccines makes WNV an ongoing public health threat. Now endemic, WNV is steadily evolving, but the contribution of positively-selected mutations to human disease remains unclear. In 2012 the second largest outbreak of human West Nile disease occurred in the U.S., with one-third of the cases happening in Texas. The outbreak was associated with groups of WNV carrying positively-selected mutations. By sequencing WNV in Texas from 2012-2015, we show that positively-selected mutations in WNV mediate increased circulation and over-wintering in the environment, which may promote increases of human disease. Additionally, we show evidence that the WNV population is still evolving new alleles. These results advance our understanding of the impact of WNV evolution to human disease, and may afford insights to the evolution of other invading flaviviruses, such as Dengue and Zika virus. Altogether, these results show that understanding the consequences of viral evolution can be harnessed towards overcoming challenges to the development of more effective therapeutics.