Bietti crystalline dystrophy (BCD) is an autosomal recessive eye disease caused by mutations in the human CYP4V2 gene. To understand the biochemistry and physiology associated with BCD, our lab has developed a “knockout” of the orthologous mouse gene, Cyp4v3. The gene targeting vector that was used to disrupt the Cyp4v3 gene incorporated a LacZ gene reporter and a neomycin antibiotic resistance gene to replace the majority (exons 2-10) of the Cyp4v3 gene. To differentiate endogenous mouse sequence from the targeting vector we designed primers to amplify by polymerase chain reaction (PCR) portions of the Cyp4v3 and LacZ genes, respectively. The experiments conducted for the LacZ primers failed to yield any PCR product when tested on samples of genomic DNA from Cyp4v3 heterozygous mice. However, we did get PCR products of the expected size from the same DNA samples for both Cyp4v3 a loading control (fatty acid binding protein-FABP). Because of the technical difficulties encountered with the LacZ primers, we designed new primers targeting the neomycin resistance gene sequence. When these new primers were tested, we were able to amplify the expected targets for all 3 genes when testing genomic DNA from a Cyp4v3 heterozygous mouse. We have developed a robust and reproducible method to genotype mice that are either Cyp4v3 +/+ (wild type), +/- (heterozygotes) or -/- (homozygous knockouts). Ongoing studies in the lab with these knockout mice are focused on characterizing the age-related progression of BCD ocular pathologies, including crystalline deposit chemical composition and defects in lipid metabolism.

Chronic inflammation in human immunodeficiency virus (HIV) infected individuals is associated with increased mortality, even in patients who are virally suppressed by antiretroviral therapy.The goal of this research is to understand how the gastrointestinal (GI) tract is damaged during early HIV/SIV infection. In many infections, neutrophils play an important part during early response to infection because of their microbial killing properties. The role of neutrophils in the establishment of HIV infection and subsequent chronic inflammation has not been examined. Our purpose was to study neutrophil supporting cytokines before and during acute simian immunodeficiency virus (SIV) infection and their potential role in the kinetics of the neutrophil response. We used SIV in the Rhesus macaque model instead of HIV so that we could obtain longitudinal samples very early after infection to observe the neutrophil kinetics. The neutrophil supporting cytokines we looked at were IL-8, IL-17, and G-CSF. We hypothesized neutrophils would decrease during acute SIV infection in peripheral blood in response to the decreasing neutrophil-supporting cytokines. We used Luminex to measure cytokine levels in the blood at each time point, and used Complete Blood Count to get blood neutrophil concentrations. After infection, neutrophils initially increased, but then 14 days post-infection there was a decrease in most of the animals. Prior to the post-infection decrease of neutrophils, we saw a decline of IL-8 levels, which may contribute to neutrophil decline. In contrast to other types of infection, we saw no increase in G-CSF or IL-17. However, we did observe increases in pro-inflammatory cytokines 14 days post-infection, and the pro-inflammatory cytokine IL-15 correlated with neutrophil decrease. These data suggest that reduced neutrophil supporting cytokines and increased pro-inflammatory cytokines may be contributing to the neutrophil decline observed in acute HIV and SIV infection.