Browsing by Author "Rojas, Leticia"

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    Capsular-defective Porphyromonas gingivalis mutant strains induce less alveolar bone resorption than W50 wild-type strain due to a decreased Th1/Th17 immune response and less osteoclast activity
    (2019) Monasterio, Gustavo; Fernandez, Baltasar; Castillo, Francisca; Rojas, Carolina; Cafferata, Emilio A.; Rojas, Leticia; Alvarez, Carla; Fernandez, Alejandra; Hernandez, Marcela; Bravo, Denisse; Vernal, Rolando
    Background Encapsulation of Porphyromonas gingivalis has been demonstrated as responsible of several host immunological changes, which have been associated with the pathogenesis of periodontitis. Using a murine model of periodontitis and two isogenic non-capsulated mutants of P. gingivalis, this study aimed to analyze whether P. gingivalis encapsulation induces more severe alveolar bone resorption, and whether this bone loss is associated with a T-helper (Th)1 and Th17-pattern of immune response. Methods Experimental periodontal infections were generated by oral inoculation with the encapsulated W50 wild-type strain or isogenic non-encapsulated Delta PG0116-PG0120 (GPA) and Delta PG0109-PG0118 (GPC) mutants of P. gingivalis. Periodontal infections induced with the encapsulated HG184 or non-encapsulated ATCC 33277 strains of P. gingivalis were used as controls. Alveolar bone resorption was analyzed using microcomputed tomography and scanning electron microscopy. The expression levels of Th1, Th2, Th17, or T regulatory-associated cytokines and RANKL, as well as the periodontal bacterial load, were quantified by quantitative polymerase chain reaction. The detection of Th1 and Th17 lymphocytes was analyzed by flow cytometry. Results In the periodontal lesions, both capsular-defective knockout mutant strains of P. gingivalis induced less alveolar bone resorption than the encapsulated W50 wild-type strain. This decreased bone loss was associated with a dismissed RANKL expression, decreased Th1- and Th17-type of cytokine expression, reduced Th1 and Th17 lymphocyte detection, and low osteoclast finding. Conclusion These data demonstrate that encapsulation of P. gingivalis plays a key role in the alveolar bone resorption induced during periodontitis, and this bone loss is associated with a Th1- and Th17-pattern of immune response triggered in the periodontal lesions.
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    FOXO1 regulates wound-healing responses in human gingival fibroblasts
    (2024) Rojas, Leticia; Tobar, Nicolas; Espinoza, Javier; Rios, Susana; Martinez, Constanza; Martinez, Jorge; Graves, Dana T.; Smith, Patricio C.
    Background and objective: Forkhead box-O 1 (FOXO1) is a transcription factor actively involved in oral wound healing at the epithelial barrier. However, less is known regarding the role of FOXO1 during the tissue repair response in the connective tissue compartment. This study explored the involvement of FOXO1 in the modulation of fibroblast activity related to wound healing. Methods: Primary cultures of human gingival fibroblasts were obtained from four healthy young donors. Myofibroblastic differentiation, collagen gel contraction, cell migration, cell spreading, and integrin activation were evaluated in the presence or absence of a FOXO1 inhibitor (AS1842856). Variations in mRNA and proteins of interest were evaluated through qRT-PCR and western blot, respectively. Distribution of actin, alpha-smooth muscle actin, and beta 1 integrin was evaluated using immunofluorescence. FOXO1 and TGF-beta 1 expression in gingival wound healing was assessed by immunohistochemistry in gingival wounds performed in C57BL/6 mice. Images were analyzed using ImageJ/Fiji. ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's post-hoc test was performed. All data are expressed as mean +/- SD. p < .05 was considered statistically significant. Results: FOXO1 inhibition caused a decrease in the expression of the myofibroblastic marker alpha-SMA along with a reduction in fibronectin, type I collagen, TGF-beta 1, and beta 1 integrin mRNA level. The FOXO1 inhibitor also caused decreases in cell migration, cell spreading, collagen gel contraction, and beta 1 integrin activation. FOXO1 and TGF-beta 1 were prominently expressed in gingival wounds in fibroblastic cells located at the wound bed. Conclusion: The present study indicates that FOXO1 plays an important role in the modulation of several wound-healing functions in gingival fibroblast. Moreover, our findings reveal an important regulatory role for FOXO1 on the differentiation of gingival myofibroblasts, the regulation of cell migration, and collagen contraction, all these functions being critical during tissue repair and fibrosis.