Comparing DC1s and DC2s Immune Response Against Cryptococcus neoformans

Elizabeth West*, Natalia Castro Lopez, Floyd Wormley Jr.
Department of Biology, Texas Christian University, Fort Worth, TX, USA

Abstract

Cryptococcus neoformans is a fungal pathogen that poses a threat to immunocompromised individuals, and there is currently no vaccine. Dendritic Cells (DCs) play a crucial role in the cell’s immune response and will be studied to determine an effective treatment. In this study, we will analyze the immune response of two groups of conventional dendritic cells (cDCs), cDC1s (CD103+, driven from GM-CSF + FLT3) and cDC2s (CD11B+, driven from GM-CSF) to determine their ability to produce a protective immune response against Cryptococcus neoformans. We grew bone marrow dendritic cells in the conditions stated above and then exposed to IFN-ɣ, cell wall extract (CWE), or both. After, we used a calcineurin (cna) knockout strain to simulate exposure to the wild-type strain, which allows us to analyze the cell’s immune response. RNA purification technique will be performed to isolate the RNA, which will then be analyzed via RT-PCR. We will analyze DC1s and DC2s responses by evaluating the transcripts, including NOS2, Arg1, and IL-2. This study will help us understand the role of DCs in the protective immune response. We hypothesize that DC1s (CD103+) will elicit a stronger Th1 response, increased by IFN-γ treatment compared to DC2s eliciting a different immune response. By comparing transcript expression levels of DC1s and DC2s, we can study the role of dendritic cell subsets in producing memory for protective immune response against Cryptococcus neoformans.

Oxidative stress is associated with the development and progression of neurodegenerative diseases, including Alzheimer’s, but there are no approved drug therapeutics that effectively target oxidative stress in Alzheimer’s. The Green Research Group has previously synthesized and reported a pyridine-containing tetra-aza macrocycle, L2, which acts as a multifunctional antioxidant agent by targeting oxidative stress directly through radical scavenging and metal ion chelation as well as catalytically through activation of the Nrf2 pathway. While multiple preliminary studies conducted on L2 have confirmed its potent antioxidant activity, its high hydrophilicity results in reduced blood-brain barrier permeability, which is a concern when designing drug therapeutics for neurodegenerative diseases. It is hypothesized that incorporating a self-immolative linker onto L2 will result in increased blood-brain barrier permeability while maintaining antioxidant activity under physiological conditions.

The shape of a drug will determine how it interacts in the body. For it to work, it must dissolve, be absorbed into the bloodstream, avoid breakdown, enter the cell and bind to its target. Each of these steps likely requires a different shape. The pharmaceutical industry has historically only focused on the shape required to bind the target. This research has identified molecules that can readily adopt multiple shapes. These ring-shaped molecules (called macrocycles) represent a new model for drug design. Usual drugs (ie ibuprofen) are small and interact with a specific target to stop a chemical reaction. Macrocycles can work by an additional mechanism. They are larger and can interfere with interactions between proteins but are still small enough to travel the body. The preparation of these macrocycles is inexpensive and quick, properties that are important for the pharmaceutical industry. This poster describes the design and synthesis of a macrocycle and an analysis of the shapes that it adopts.

Every 65 seconds, someone develops Alzheimer's disease, which is the seventh leading cause of death in the United States. A major barrier to potential therapeutics is the permeability of these molecules across the blood-brain barrier. We have developed small molecules with strong reactivity to combat the oxidative stress known to cause Alzheimer’s disease. However, the permeability is less than ideal. As a result, my goal is to produce a molecule that has enhanced permeability but retains the reactivity of the parent molecules. To achieve this, the BOILED-Egg model assessed different derivatives of our parent molecule, Py2N2. This model showed the differences in lipophilicity among different Py2N2 compounds and how they impact permeability into the blood-brain barrier and gastrointestinal tract. Background information on our parent molecule and its function regarding Alzheimer's development will be outlined to give a scope of what these compounds can target and how they function. Compounds with high lipophilicity reflected in the model will have schemes of synthetic synthesis for future directions.

Perfluoroalkyl substances (PFAS), known as "forever chemicals", are ubiquitous environmental contaminants whose remarkable persistence poses significant risks to human health and ecosystems. Thus, it is important to develop analytical assays to determine PFAS concentrations based on widely accessible, readily available instrumentation, such as UV-VIS spectrophotometry. Tetrasodium tetraphenylporphyrintetrasulfonate (TPPS) is a water-soluble porphyrin known for its spectrophotometric property in water. It is also known that TPPS binds to the protein bovine serum albumin (BSA). We investigated the effect of BSA on the absorption spectrum of TPPS and how PFAS presence impacts BSA-TPPS interaction in water. Interestingly, we found that BSA induces TPPS precipitation. As BSA concentration increases, TPPS solubility first dramatically decreases, then increases, ultimately leading to the formation of homogeneous solutions at relatively high BSA concentration. Furthermore, addition of two different PFAS, sodium perfluorohexanoate and potassium perfluorobutanesulfonate salts, to homogeneous BSA-TPPS mixtures appreciably alter TPPS spectra. Our results show that these mixtures can be used to produce calibration curves relevant to the determination of PFAS concentrations in water.