Examinando por Autor "Hernández, M."
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Ítem CpG Single-Site Methylation Regulates TLR2 Expression in Proinflammatory PBMCs From Apical Periodontitis Individuals(Frontiers Media S.A., 2022-03) Bordagaray, M.; Fernández, A.; Astorga, J.; Garrido, M.; Hernández, P.; Chaparro, A.; Lira, M.; Gebicke-Haerter, P.; Hernández, M.Apical periodontitis (AP) is a common oral disease caused by the inflammatory destruction of the periapical tissues due to the infection of the root canal system of the tooth. It also contributes to systemic bacterial translocation, where peripheric mononuclear blood cells (PBMCs) can act as carriers. Toll-like receptor (TLR) 2 mediates the response to infection and activates inflammatory responses. DNA methylation can be induced by bacteria and contributes to the modulation of this response. Despite the evidence that supports the participation of PBMCs in immune-inflammatory disorders, the inflammatory profile and epigenetic regulatory mechanisms of PBMCs in AP individuals are unknown. Aim: To determine TLR2 gene methylation and inflammatory profiles of PBMCs in AP. Methods: Cross-sectional exploratory study. Otherwise, healthy individuals with AP (n=27) and controls (n=30) were included. PMBCs were isolated by a Ficoll gradient, cultured for 24 hours, and both RNA and DNA were extracted. DNA was bisulfite-treated, and specific sites at the promoter region of the TLR2 gene were amplified by qPCR using validated primers. To verify its amplification, agarose gels were performed. Then, the PCR product was sequenced. mRNA expression of TLR2 was determined by qPCR. The soluble levels of 105 inflammatory mediators were first explored with Proteome Profiler Human Cytokine Array Kit. Consequently, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-6Rα, IL-1β, and IL-12p70 levels were measured by Multiplex assay. Results: PBMCs from individuals with AP demonstrated a proinflammatory profile showing higher soluble levels of TNF-α, IL-6, and IL-1β compared to controls (p<0.05). Higher TLR2 expression and higher global methylation pattern of the promoter region of the gene were found in AP compared to controls (p<0.05). The CpGs single-sites at positions -166 and -146 were completely methylated, while the site -102 was totally unmethylated, independently of the presence of AP. DNA methylation of CpG single-sites in positions -77 and +24 was positively associated with TLR2 expression. Conclusions: PBMCs from AP subjects show a hyperinflammatory phenotype and TLR2 upregulation in association with single CpG-sites’ methylation from the TLR2 gene promoter, thereby contributing to a sustained systemic inflammatory load in individuals with periapical endodontic diseases.Ítem Fasciola hepatica coinfection modifies the morphological and immunological features of Echinococcus granulosus cysts in cattle(BioMed Central Ltd., 2020-06) Hidalgo, C.; Stoore, C.; Hernández, M.; Paredes, R.Polyparasitism occurs when animals harbour multiple parasites concomitantly. It is a common occurrence but is generally understudied in wild and domestic animals. Fasciola hepatica and Echinococcus granulosus, which are helminths of ungulates, frequently coinfect cattle. The effects of this particular type of polyparasitism are not well documented. The metacestode of Echinococcus granulosus is surrounded by the adventitial layer, which constitutes the host immune response to the parasite. This layer in cattle is produced by a granulomatous reaction and is involved in echinococcal cyst (EC) fertility. Due to the systemic immune-modulating abilities of Fasciola hepatica, coinfection possibly generates a favourable environment for EC growth. A total of 203 Echinococcus granulosus sensu stricto cysts were found in 82 cattle, of which 42 ECs were found in 31 animals coinfected with Fasciola hepatica. The overall infection intensity was 3 cysts per animal. Coinfection with Fasciola hepatica decreased the mean infection intensity to 1.4 cysts per animal. Regarding EC size, coinfection resulted in smaller ECs (15.91 vs 22.09 mm), especially for infertile lung cysts. The adventitial layer of ECs in coinfected animals lacked lymphoid follicles and palisading macrophages, which are generally hallmarks of the granulomatous immune response. The ECs in coinfected animals had organized laminated layers, whereas those in animals without coinfection did not. Although coinfection was not statistically associated with EC fertility, we did not find fertile cysts in the livers of coinfected animals. We concluded that coinfection with Fasciola hepatica and Echinococcus granulosus has a detrimental effect on ECs, particularly infertile cysts. © 2020 The Author(s).Ítem First description of echinococcus ortleppi and cystic echinococcosis infection status in Chile(Public Library of Science, 2018-05) Corrêa, F.; Stoore, C.; Horlacher, P.; Jiménez, M.; Hidalgo, C.; Alvarez Rojas, C.A.; Barros, G.F.; Ferreira, H.B.; Hernández, M.; Cabrera, G.; Paredes, R.Cystic echinococcosis (CE), a parasitic disease caused by the cestode Echinococcus granulosus sensu lato (s.l.), is a worldwide zoonotic infection. Although endemic in Chile, information on the molecular characteristics of CE in livestock remains scarce. Therefore we aimed to describe the status of infection with E. granulosus s.l. in cattle from central Chile and also to contribute to the study of the molecular epidemiology of this parasite. According to our results, the prevalence of CE is 18.84% in cattle, similar to previous reports from Chile, suggesting that the prevalence in Santiago Metropolitan area has not changed in the last 30 years. Most of the cysts were found only in lungs (51%), followed by concurrent infection in liver and lungs (30%), and only liver (19%). Molecular characterization of the genetic diversity and population structure of E. granulosus s.l. from cattle in central Chile was performed using a section of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. E. granulosus sensu stricto (s.s.) (G1-G3 genotypes) was confirmed by RFLP-PCR to be the dominant species affecting cattle (284 samples/290 samples); we also report for the first time in Chile the presence of E. ortleppi (G5 genotype) (2 samples/61 samples). The Chilean E. granulosus s.s. parsimony network displayed 1 main haplotype. Additional studies using isolates from many locations across Chile and different intermediate hosts will provide more data on the molecular structure of E. granulosus s.s. within this region. Likewise, investigations of the importance of E. ortleppi in human infection in Chile deserve future attention. © 2018 Corrêa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Ítem Levels of the interleukins 17A, 22, and 23 and the S100 protein family in the gingival crevicular fluid of psoriatic patients with or without periodontitis(Sociedade Brasileira de Dermatologia, 2021) Jiménez, C.; Carvajal, D.; Hernández, M.; Valenzuela, F.; Astorga, J.; Fernández, A.Background Psoriasis and periodontitis are immunologically mediated chronic inflammatory diseases. Epidemiologic evidence has linked both; however, the change of markers in gingival crevicular fluid has been poorly evaluated. Objective To evaluate the levels of IL-17A, IL-22, IL-23, S100A7, S100A8, and S100A9 in gingival crevicular fluid of psoriatic and healthy subjects with and without periodontitis and their relations to psoriasis severity. Methods Cross-sectional study. Sample comprised the following groups: healthy controls without periodontitis or with mild periodontitis (n = 21), healthy controls with moderate or severe periodontitis (n = 18), individuals with psoriasis without or mild periodontitis (n = 11), and individuals with psoriasis and moderate or severe periodontitis (n = 32). Levels of IL-17A, IL-22, IL-23, S100A8, and S100A9 were determined by multiplex assay and S100A7 was measured by ELISA. Results No inter-group differences in the levels of IL-17A, IL-22, IL-23, and S100A7 were found. S100A8 levels were higher in psoriatic patients than controls (p < 0.05). S100A8 was positively correlated with psoriasis severity in the group with psoriasis (p < 0.05). S100A9 exceeded the detection limits. Study limitations This pilot study presents a small sample size. Conclusions The concentrations of S100A8 were highest in psoriatic patients regardless of periodontal health/status. S100A8 was associated with the severity of psoriasis. The concentrations of interleukins and S100A7 were similar in psoriatic patients with or without periodontitis vs. healthy controls.Ítem Subcellular location of Piscirickettsia salmonis heat shock protein 60 (Hsp60) chaperone by using immunogold labeling and proteomic analysis(MDPI AG, 2020) Oliver, C.; Sánchez, P.; Valenzuela, K.; Hernández, M.; Pontigo, J.; Rauch, M.; Garduño, R.; Avendaño-Herrera, R.; Yáñez, A.Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by the T4SS, T3SS, and T6SS, with negative results. These data support the hypothesis that small amounts of Hsp60 must reach the bacterial cell surface in a manner probably not mediated by currently characterized secretion systems, and that they remain biologically active during P. salmonis infection, possibly mediating adherence and (or) invasion.Ítem Systemic and Extraradicular Bacterial Translocation in Apical Periodontitis(Frontiers Media S.A., 2021-03) Bordagaray, M.J.; Fernández, A.; Garrido, M.; Astorga, J.; Hoare, A.; Hernández, M.Apical periodontitis is an inflammatory disease of microbial etiology. It has been suggested that endodontic bacterial DNA might translocate to distant organs via blood vessels, but no studies have been conducted. We aimed first to explore overall extraradicular infection, as well as specifically by Porphyromonas spp; and their potential to translocate from infected root canals to blood through peripheral blood mononuclear cells. In this cross-sectional study, healthy individuals with and without a diagnosis of apical periodontitis with an associated apical lesion of endodontic origin (both, symptomatic and asymptomatic) were included. Apical lesions (N=64) were collected from volunteers with an indication of tooth extraction. Intracanal samples (N=39) and respective peripheral blood mononuclear cells from apical periodontitis (n=14) individuals with an indication of endodontic treatment, as well as from healthy individuals (n=14) were collected. The detection frequencies and loads (DNA copies/mg or DNA copies/μL) of total bacteria, Porphyromonas endodontalis and Porphyromonas gingivalis were measured by qPCR. In apical lesions, the detection frequencies (%) and median bacterial loads (DNA copies/mg) respectively were 70.8% and 4521.6 for total bacteria; 21.5% and 1789.7 for Porphyromonas endodontalis; and 18.4% and 1493.9 for Porphyromonas gingivalis. In intracanal exudates, the detection frequencies and median bacterial loads respectively were 100% and 21089.2 (DNA copies/μL) for total bacteria, 41% and 8263.9 for Porphyromonas endodontalis; and 20.5%, median 12538.9 for Porphyromonas gingivalis. Finally, bacteria were detected in all samples of peripheral blood mononuclear cells including apical periodontitis and healthy groups, though total bacterial loads (median DNA copies/μL) were significantly higher in apical periodontitis (953.6) compared to controls (300.7), p<0.05. Porphyromonas endodontalis was equally detected in both groups (50%), but its bacterial load tended to be higher in apical periodontitis (262.3) than controls (158.8), p>0.05; Porphyromonas gingivalis was not detected. Bacteria and specifically Porphyromonas spp. were frequently detected in endodontic canals and apical lesions. Also, total bacteria and Porphyromonas endodontalis DNA were detected in peripheral blood mononuclear cells, supporting their plausible role in bacterial systemic translocation. © Copyright © 2021 Bordagaray, Fernández, Garrido, Astorga, Hoare and Hernández.Ítem The Incubation of 13α,17-Dihydroxystemodane with Cephalosporium aphidicola(MDPI AG, 2012-02) Fraga, B.; Guillermo, R.; Hernández, M.; Chamy, M.; Garbarino, J.The biotransformation of 13α,17-dihydroxystemodane (3) with the fungus Cephalosporium aphidicola afforded 13α,17,18-trihydroxystemodane (4), 3β,13α,17-trihydroxystemodane (5), 13α,17-dihydroxy- stemodan-18-oic acid (6), 3β,11β,13α,17-tetrahydroxystemodane (7), 11β,13α,17,18-tetrahydroxystemodane (8) and 3β,13α, 17,18-tetrahydroxystemodane (9). The hydroxylation at C-18 of the substrate points to a biosynthetically-directed transformation, because aphidicolin (2) is hydroxylated at this carbon. However, the C-3(β) and C-11(β) hydroxylations seem to indicate a xenobiotic biotransformation.