Examinando por Autor "Schinnerling, Katina"
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Ítem Efecto de Prevotella copri sobre la viabilidad, activación y diferenciación de monocitos y macrófagos(Universidad Andrés Bello, 2023) Muñoz Mora, Karim Fernanda; Schinnerling, Katina; Facultad de Ciencias de la VidaLa microbiota intestinal es una comunidad de diversos géneros bacterianos comensales que interactúan con el individuo en el que habitan, causando impacto en el sistema inmune. Sin embargo, su disbiosis se asocia con el desarrollo de patologías inflamatorias como la Artritis Reumatoide (AR), enfermedad autoinmune que causa la degradación de cartílago y material óseo en las articulaciones sinoviales. Varios estudios han relacionado la AR a la especie Prevotella copri (P. copri) encontrándose sobreabundante en la microbiota intestinal de pacientes con AR temprana y pre-clínica. Se ha demostrado que P. copri promueve el desarrollo de artritis experimental en ratones SKG e induce un aumento de linfocitos Th17. Además, P. copri reduce el grosor de la capa protectora de la mucosa intestinal. Esto sugiere la participación de P. copri en el desarrollo y progresión de AR. Para dilucidar el mecanismo por el cual P. copri podría contribuir a la inmunopatogénesis de la AR, este proyecto se enfoca en investigar los efectos de P. copri sobre monocitos y macrófagos humanos. Dado que se ha reportado que pacientes con AR activa presentan un sesgo hacia macrófagos proinflamatorios M1, se hipotetiza que P. copri reduce la viabilidad, promueve la activación y diferenciación de monocitos y macrófagos hacia un perfil M1 in vitro. El objetivo general es evaluar el efecto de P. copri sobre la viabilidad, activación y diferenciación de monocitos y macrófagos in vitro. Como modelo de monocitos se empleó la línea celular THP1, que puede diferenciarse a macrófagos al ser expuesta a PMA. Se determinó el efecto de P. copri sobre la viabilidad de monocitos y macrófagos in vitro, mediante marcaje con Anexina V y una sonda de viabilidad mediante citometría de flujo. Luego, se evaluó el efecto de P. copri sobre la activación y diferenciación de monocitos y macrófagos in vitro, mediante el análisis de marcadores para M1/M2 y del perfil de citoquinas por citometría de flujo. Este estudio contribuye al conocimiento sobre la interacción entre P. copri y el sistema inmune innato, particularmente los monocitos y macrófagos, en la inmunopatogénesis de AR, que podrían asociarse al desarrollo y propagación de inflamación crónica en AR.Ítem Estudio de internalización y mecanismos de endocitosis de las vesículas de membrana externa de Prevotella copri en macrófago(Universidad Andrés Bello, 2024) Chávez Villacreses, Karissa Jamileth; Schinnerling, Katina; Soto Ramírez, Jorge; Facultad de Ciencias de la VidaLa Artritis Reumatoide (AR) es una enfermedad autoinmune por etapas en donde la inflamación crónica generada en tejido sinovial puede causar deformidad en distintas articulaciones como manos, rodillas, muñecas, pies, entre otros. Se describe como enfermedad autoinmune debido a la presencia de autoanticuerpos como el factor reumatoide (FR) y anticuerpos anti-peptidos citrulinadas (ACPA). Hay distintos factores genéticos y ambientales que pueden influenciar en el padecimiento de AR como la dieta occidental, fumar, la sobreabundancia o disminución de algún microorganismo en la microbiota intestinal de la persona. Distintos estudios han encontrado directa relación entre la disbiosis en la microbiota gastrointestinal y la inflamación dada en el tejido sinovial de las articulaciones tanto en pacientes como en modelos murinos para AR. La bacteria anaerobia Gram negativo Prevotella copri se encuentra normalmente en la microbiota de las personas, pero en situaciones de AR se encuentra en mayor abundancia. Se ha reportado que el péptido de P. copri denominado Pc-p27 es presentado por la molécula MHC-II de las células presentadoras de antígenos, como los macrófagos, desencadenando respuesta de células T y la producción de anticuerpos en pacientes con AR. Por otra parte, se encuentran las vesículas de membrana externa (OMV) que desempeñan un papel fundamental en la interacción bacteria-huésped gracias a que presentan estructuras de la bacteria parental como LPS, proteínas de membrana, peptidoglucanos, etc. El principal objetivo que tiene este proyecto es evaluar el mecanismo de endocitosis utilizado por los macrófagos para internalizar las OMVs de Prevotella copri. Se realizó el cultivo anaerobio de P. copri para la obtención de sus OMVs y posterior incubación en macrófagos primarios derivados de PBMC adquiriendo imágenes mediante microscopía confocal. También se realizó estimulación con OMVs en células RAW264.7 para realizar cinéticas de internalización e inhibición de vías de endocitosis mediante inhibidores farmacológicos. Se obtuvieron imágenes con señal fluorescente de las OMVs-DiO en macrófagos primarios, cinética internalización de las OMVs durante 2 horas, internalización de OMVs-DiO dependiente de concentración, además de sugerirse que la vía de internalización de las vesículas fue la endocitosis mediada por clatrinas. La relevancia de este proyecto fue comprender el mecanismo por el cual células presentadoras de antígenos como los macrófagos pueden internalizar las OMVs pertenecientes a una bacteria relacionada actualmente con el desarrollo de la artritis reumatoide.Ítem Human metapneumovirus respiratory infection affects both innate and adaptive intestinal immunity(Frontiers Media SA, 2024) Sepúlveda-Alfaro, Javiera; Catalán, Eduardo A.; Vallejos, Omar P.; Ramos-Tapia, Ignacio; Madrid-Muñoz, Cristóbal; Mendoza-León, María J.; Suazo, Isidora D.; Rivera-Asin, Elizabeth; Silva, Pedro H.; Alvarez-Mardones, Oscar; Castillo-Godoy, Daniela P.; Riedel, Claudia A.; Schinnerling, Katina; Ugalde, Juan A.; Soto, Jorge A.; Bueno, Susan M.; Kalergis, Alexis M.; Melo-Gonzalez, FelipeIntroduction: Respiratory infections are one of the leading causes of morbidity and mortality worldwide, mainly in children, immunocompromised people, and the elderly. Several respiratory viruses can induce intestinal inflammation and alterations in intestinal microbiota composition. Human metapneumovirus (HMPV) is one of the major respiratory viruses contributing to infant mortality in children under 5 years of age worldwide, and the effect of this infection at the gut level has not been studied. Methods: Here, we evaluated the distal effects of HMPV infection on intestinal microbiota and inflammation in a murine model, analyzing several post-infection times (days 1, 3, and 5). Six to eight-week-old C57BL/6 mice were infected intranasally with HMPV, and mice inoculated with a non-infectious supernatant (Mock) were used as a control group. Results: We did not detect HMPV viral load in the intestine, but we observed significant changes in the transcription of IFN-γ in the colon, analyzed by qPCR, at day 1 post-infection as compared to the control group. Furthermore, we analyzed the frequencies of different innate and adaptive immune cells in the colonic lamina propria, using flow cytometry. The frequency of monocyte populations was altered in the colon of HMPV -infected mice at days 1 and 3, with no significant difference from control mice at day 5 post-infection. Moreover, colonic CD8+ T cells and memory precursor effector CD8+ T cells were significantly increased in HMPV-infected mice at day 5, suggesting that HMPV may also alter intestinal adaptive immunity. Additionally, we did not find alterations in antimicrobial peptide expression, the frequency of colonic IgA+ plasma cells, and levels of fecal IgA. Some minor alterations in the fecal microbiota composition of HMPV -infected mice were detected using 16s rRNA sequencing. However, no significant differences were found in β-diversity and relative abundance at the genus level. Discussion: To our knowledge, this is the first report describing the alterations in intestinal immunity following respiratory infection with HMPV infection. These effects do not seem to be mediated by direct viral infection in the intestinal tract. Our results indicate that HMPV can affect colonic innate and adaptive immunity but does not significantly alter the microbiota composition, and further research is required to understand the mechanisms inducing these distal effects in the intestine.Ítem Oral treatment of Whipple's disease with doxycycline and hydroxychloroquine versus intravenous therapy with ceftriaxone followed by oral trimethoprim–sulfamethoxazole in Germany: a phase 2/3, prospective, open-label, randomised, controlled, non-inferiority trial(The Lancet Infectious Diseases, 2025, 2025) Moos, Verena; Krüger, Justina; Allers, Kristina; Moter, Annette; Kikhney, Judith; Kühl, Anja A; Loddenkemper, Christoph; Stroux, Andrea; Schinnerling, Katina; Schneider, ThomasBackground: Previous studies have shown that intravenous ceftriaxone or meropenem for 14 days, followed by oral trimethoprim–sulfamethoxazole for 1 year, cures 98% of people with Whipple's disease. However, intravenous therapy requires hospitalisation and carries risks for treatment-associated complications. The aim of this study was to investigate whether oral-only treatment for Whipple's disease is non-inferior to intravenous therapy. Methods: This phase 2/3, prospective, open-label, randomised, controlled, non-inferiority trial enrolled individuals aged 18 years or older with confirmed Whipple's disease from across Germany who had received treatment for less than 1 month at Charité–Universitätsmedizin Berlin. Participants were randomly assigned (1:1) with block randomisation to receive either intravenous ceftriaxone (2 g once per day) for 14 days, followed by oral trimethoprim–sulfamethoxazole (960 mg twice per day) for 12 months, or oral doxycycline (100 mg twice per day) plus hydroxychloroquine (200 mg twice per day) for 12 months. Ten participants who had already received intravenous ceftriaxone were non-randomly assigned to the intravenous treatment group. Participants in the oral-only treatment group were PCR-positive for Tropheryma whipplei in cerebrospinal fluid received trimethoprim–sulfamethoxazole (960 mg five times per day) until clearance. The primary outcome was complete clinical remission without recurrence during the observation period of 24 months, assessed in the intention-to-treat (ITT) population. The prespecified non-inferiority margin was –18%. Safety was a secondary endpoint, assessed in the ITT population. The study was registered with the EU Clinical Trials Register, EudraCT 2008–003951–54, and is completed. Findings: Between May 26, 2010, and Oct 30, 2018, we screened 310 individuals and enrolled 64 participants in the study. After exclusion of four individuals whose diagnosis was not confirmed, 31 participants were assigned to the intravenous treatment group and 29 to the oral-only treatment group. By ITT, 25 (81%) of 31 participants in the intravenous treatment group and 28 (97%) of 29 participants in the oral-only treatment group had complete clinical remission without recurrence. The risk difference was 15·9 percentage points (95% CI –1·2 to 33·1), with the lower bound of the 95% CI above our non-inferiority margin of –18%. A post-hoc per-protocol analysis confirmed the non-inferiority of oral-only treatment. No participant relapsed, but two participants in the intravenous treatment group died from nosocomial infections. Serious adverse events occurred in 13 (42%) of 31 participants in the intravenous treatment group and eight (28%) of 29 participants in the oral-only treatment group, but this difference was not statistically significant (p=0·244). Interpretation: Oral-only treatment of Whipple's disease was safe and non-inferior to sequential intravenous–oral treatment. Oral treatment facilitates patient management and might reduce hospital-acquired treatment complications and costs. Funding: German Research Foundation and the Robert Koch Institute. Translation: For the German translation of the abstract see Supplementary Materials section. © 2025 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC 4.0 licenseÍtem Uptake of Tropheryma whipplei by Intestinal Epithelia(MDPI, 2023-04) Friebel, Julián; Schinnerling, Katina; Weigt, Kathleen; Heldt, Claudia; Fromm, Anja; Bojarski, Christian; Siegmund, Britta; Epple, Hans-Jörg; Kikhney, Judith; Moter, Annette; Schneider, Thomas; Schulzke, Jörg D.Background: Tropheryma whipplei (TW) can cause different pathologies, e.g., Whipple’s disease and transient gastroenteritis. The mechanism by which the bacteria pass the intestinal epithelial barrier, and the mechanism of TW-induced gastroenteritis are currently unknown. Methods: Using ex vivo disease models comprising human duodenal mucosa exposed to TW in Ussing chambers, various intestinal epithelial cell (IEC) cultures exposed to TW and a macrophage/IEC coculture model served to characterize endocytic uptake mechanisms and barrier function. Results: TW exposed ex vivo to human small intestinal mucosae is capable of autonomously entering IECs, thereby invading the mucosa. Using dominant-negative mutants, TW uptake was shown to be dynamin- and caveolin-dependent but independent of clathrin-mediated endocytosis. Complementary inhibitor experiments suggested a role for the activation of the Ras/Rac1 pathway and actin polymerization. TW-invaded IECs underwent apoptosis, thereby causing an epithelial barrier defect, and were subsequently subject to phagocytosis by macrophages. Conclusions: TW enters epithelia via an actin-, dynamin-, caveolin-, and Ras-Rac1-dependent endocytosis mechanism and consecutively causes IEC apoptosis primarily in IECs invaded by multiple TW bacteria. This results in a barrier leak. Moreover, we propose that TW-packed IECs can be subject to phagocytic uptake by macrophages, thereby opening a potential entry point of TW into intestinal macrophages.