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Browsing by Author "Magee, Christianne, committee member"

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    Establishment and characterization of day 30 equine chorionic girdle and allantochorion cell lines
    (Colorado State University. Libraries, 2019) Salman, Saleh M., author; Bruemmer, Jason E., advisor; Bouma, Gerrit, advisor; Magee, Christianne, committee member; Pinedo, Pablo, committee member; Winger, Quinton, committee member
    Establishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. A human resistant telomerase reverse transcriptase (hTERT) lentivirus was utilized to establish stable equine embryonic cell lines. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast are the allantochorion (AC). Embryonic CG cells are unique to horses compared to other farm animals' embryos. The CG cells are the predecessor of endometrial cups (EC) that differentiate, proliferate, and invade the endometrium by day 38 of pregnancy, yet morphologically, both have similar characteristics supporting the fetal origin for EC. The CG cells have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study has three objectives: 1) establishing a stable cell line from day 30 CG cells and AC using lentivirus encoding hTERT; 2) Characterization of day 30 CG cells and AC cell morphology and expression of eCG alpha (eCGα) and beta (eCGβ) subunits, major histocompatibility complex class II (MHC II), and Kisspeptin receptor (KISS1R) in CG and AC cells; 3) investigating eCG protein production in vitro from day 30 CG and AC cells. Reverse transcriptase (RT) PCR was used to study gene expression in cells and radioimmunoassay (RIA) was used to investigate protein presence in the media. We established a hygromycin-resistant day 30 CG and AC cell lines that express eCGα, eCGβ, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages, 10 of which were cultured after the lentiviral infection steps. Also, we characterized CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGα and eCGβ subunits are expressed by both day 30 CG and AC cells, but MHC II and KISS1R genes were not expressed in either of cells. Moreover, RIA results showed that day 30 CG cells did produce eCG protein in vitro earlier than what previous literature has shown. However, day 30 AC cells did not produce eCG protein in vitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection.
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    Using a precision-cut lung slice co-culture paradigm to increase T-cell populations and model infection ex vivo
    (Colorado State University. Libraries, 2024) Ehrlich, Alexis T., author; Tobet, Stuart, advisor; Perera, Rushika, committee member; Magee, Christianne, committee member
    Precision cut lung slices (PCLS) bridge a gap between in vivo and in vitro studies by maintaining anatomical organization with structural integrity and intercellular signaling pathways. Applications of PCLS have included the modeling of inflammatory lung diseases, metabolism studies, and drug development. In the lungs, immune responses are carried out by a network of T- and B- cells, the latter of which are resident. The limited resident T-cell population of the lung diminishes accurate representations of pathogen response capacity in PCLS. Addressing this, we set out to increase pulmonary T-cell populations ex vivo. We hypothesized that thymus and bone marrow-derived T-cells would work synergistically to populate the lung in co-culture experiments. A murine organotypic lung co-culture model was developed and characterized for tissue health and T-cell recruitment over 3 days ex vivo using adult neurobasal media with 4 mM glucose + 2% B27 supplement. Lung slices were cultured independently, with bone marrow, thymus, or both. Immune colonization of the lung was assessed using immunohistochemistry for CD3+ T-cells, CD19+ B-cells and ACK2+ cells. Cells were counted in alveolar and airway spaces after 3 days of culture. Our results demonstrate that lung co-cultured with thymus and bone significantly increased T-cell populations ex vivo, whereas lung co-cultured with thymus or bone alone did not significantly alter T-cell counts. Additionally, B-cells and C-Kit+ cell populations were not influenced by the culture paradigm. Using this paradigm, we went on to explore this lung co-culture paradigm when stimulated by an immune modulatory agent – LPS- and when an active lung infection is present using Pseudomonas aeruginosa. Lung and lung co-cultures had increases in T-cell counts after immune stimulation and infection. Additionally, the co-cultures further increased T-cell counts after treatment. More strikingly, the co-cultures influenced the degree of bacterial infection in the lung slices without altering the B-cell populations among cultures. Viral infections are also common pathogens that affect the lungs, so we examined then examined the ability of viral pathogens to infect precision cut lung slices. As we did not get an infection with live virus, we explored the effect of a viral mimic – Resiquimod- on lung co-cultures on the immune responses. Resiquimod and the co-cultures are both able to increase T-cell populations in lung slices ex vivo. These results suggest that the increased T-cell population corresponding with thymus and bone marrow co-culture could be a result of cell-cell interaction or the secretion of growth factors. Cell secretions or growth factor release could stimulate thymic secretion of T-cells or could stimulate T-cell proliferation in the lung, suggesting that co-culture with thymus and bone marrow can elicit a T-cell response ex vivo. T-cells are necessary for host-pathogen immune responses, most commonly by CD8+ T-cells but there are other populations of T-cells. Although there are limitations to the use of lung slices in infection studies, the results of the co-culture and increasing T-cells is a promising step to studying pathogen response capacity in the future ex vivo.