Immunomodulatory activity of itraconazole in lung

Purpose: To evaluate the in vivo effects of ITC as an immunomodulator on various immunological mediators, including cytokines, chemokines and growth factors in pulmonary homogenates.Methods: Two experimental groups consisting of 25 BALB/c mice each were used: (a) PBS-treated mice and (b) ITC-treated mice (1 mg/mouse/day). The animals were treated daily via gavage for 8 weeks. Five mice from each group were sacrificed at 0, 4, 8, and 12 weeks and cytokine levels assessed in the supernatants of lung macerates using the Bio-Plex system. Five lungs from each experimental group, after 8 weeks of treatment, were used for histopathological analysis.Results: Compared with the control group, ITC-treated mice showed significant changes in the pulmonary levels of 50 % of the molecules evaluated: IL-4, IL-10, IL-12p70, IL-13, TNF-α, eotaxin, MIP- 1α, MIP-2, and LIF were significantly upregulated. In contrast, IL-1β, IL-15, IL-2, IL-1α, and VEGF were downregulated.Conclusion: Although histopathological examination did not show changes in lung cell infiltrates, ITC exerted a marked immunomodulatory effect at the pulmonary level in healthy BALB/c mice.Keywords: Immunomodulation, Itraconazole, Antifungal drug, Histopathological examinatio

Estandarización y validación de un método de cromatografía líquida de alta eficiencia con detector de arreglo de diodos (HPLC-DAD) para la determinación de niveles sanguíneos de voriconazol

Introduction. A specialized service for the determination of antifungal blood levels is not available in Colombia, this service is essential for the proper follow-up of the antifungal therapy.Objective. The aim of this study was to standardize and validate a simple, sensitive, and specific protocol, based on High Performance Liquid Chromatography with Diode Array Detector (HPLC-DAD) for the quantification of voriconazole blood levels. Materials and methods. An Agilent HPLC-series-1200 equipment with UV-DAD detector was used. Analytical column-Eclipse XDB-C18 and pre- column Eclipse-XDB-C18, Agilent were also used. We used voriconazole as the primary control, and posaconazole as an internal control. The validation was done following the Food and Drug Administration (FDA) recommendations.Results. Best chromatographic conditions were: Column temperature of 25°C, UV variable wavelength detectors (VWD) detection at 256 nm for voriconazole, and 261 nm for Posaconazole (internal standard), 50 μL of injection volume, flow of volume 0,8mL/min, time of run of 10 min, mobile phase of acetonitrile:water (60:40). Finally, retention times were 3.13 and 5.16 min for the voriconazole and posaconazole, respectively. Range of quantification ranging from 0.125 μg/mL to 16 μg/mL. Conclusion. The selectivity and chromatographic purity of the obtained signal as well as the limits of detection and quantification standardized make this method an excellent tool for therapeutic monitoring of patients treated with voriconazole.Introducción. Hasta la fecha en Colombia no contábamos con un servicio especializado de medición de niveles séricos de antifúngicos, procedimiento esencial para el adecuado manejo del tratamiento de las Infecciones Fúngicas Invasoras (IFI). Objetivo. Estandarizar y validar un protocolo, simple, sensible y específico, basado en Cromatografía Líquida de Alta Eficiencia con detector de arreglo de diodos (HPLC-DAD) para la cuantificación de los niveles séricos de voriconazol. Materiales y métodos. Se usó un equipo HPLC-Agilent, serie-1200, con detector UV-DAD columna analítica Eclipse-XDB-C18 y una pre-columna Eclipse-XDB-C18, ambas de la marca Agilent. Como control primario se utilizó voriconazol, y como control interno posaconazol. La validación se hizo cumpliendo todos los criterios de aceptación de los parámetros recomendados por la Food and Drug Administration (FDA). Resultados. Las mejores condiciones cromatográficas se obtuvieron bajo los siguientes parámetros: temperatura de la columna de 25°C, detección UV-VWD de 261 nm, volumen de inyección de 50 μL, flujo de 0,8mL/min y un tiempo de corrido de 10 min. La fase móvil usada fue acetonitrilo:agua (40:60), obteniendo finalmente unos tiempos de retención de 3,13 y 5,16 min para el voriconazol y posaconazol respectivamente. El rango de cuantificación fue desde 0,125 μg/mL hasta 16 μg/mL. Conclusiones. La selectividad y pureza de la señal cromatográfica obtenida, así como los límites de detección y cuantificación estandarizados, hacen de esta metodología una excelente herramienta para el seguimiento terapéutico de los pacientes bajo tratamiento o profilaxis con voriconazol

Structural and Topographic Dynamics of Pulmonary Histopathology and Local Cytokine Profiles in Paracoccidioides brasiliensis Conidia-Infected Mice

Paracoccidioidomycosis (PCM), an endemic fungal infection of pulmonary origin resulting in severe disseminated disease, occurs in rural areas of most South American countries and presents several clinical forms. The infection is acquired by inhalation of specific fungal propagules, called conidia. Considering the difficulties encountered when studying the infection in humans, this work was done in mice infected by inhalation of infective fungal conidia thus mimicking the human natural infection. The lungs of mice were sequentially studied by histopathological and multiplex cytokine methods from 2 h to 16 weeks after infection to verify the course of the disease. The mycosis presented different morphologic aspects during the course of time, affecting several pulmonary compartments. Otherwise and based on the analysis of 30 cytokines, the immune response also showed heterogeneous responses, which were up or down regulated depending on the time of infection. By recognizing the different stages that correspond to the evolution of pulmonary lesions, the severity (benign, chronic or fibrotic) of the disease could be predicted and the probable prognosis of the illness be inferred

Antifungal Encapsulated into Ligand-Functionalized Nanoparticles with High Specificity for Macrophages

Infectious diseases caused by intracellular microorganisms such as Histoplasma capsulatum represent a significant challenge worldwide. Drug encapsulation into functionalized nanoparticles (NPs) is a valuable alternative to improving drug solubility and bioavailability, preventing undesirable interactions and drug degradation, and reaching the specific therapeutic target with lower doses. This work reports on Itraconazole (ITZ) encapsulated into core-shell-like polymeric NPs and functionalized with anti-F4/80 antibodies for their targeted and controlled release into macrophages. Uptake assay on co-culture showed significant differences between the uptake of functionalized and bare NPs, higher with functionalized NPs. In vitro assays showed that F4/80-NPs with 0.007 µg/mL of encapsulated ITZ eliminated the H. capsulatum fungus in co-culture with macrophages effectively compared to the bare NPs, without any cytotoxic effect on macrophages after 24 h interaction. Furthermore, encapsulated ITZ modulated the gene expression of anti and pro-inflammatory cytokines (IL-1, INF-Y, IL-6 and IL-10) on macrophages. Additionally, the anti-F4/80 antibody-coating enhanced natural and adequate antifungal response in the cells, exerting a synergistic effect that prevented the growth of the fungus at the intracellular level. Functionalized NPs can potentially improve macrophage-targeted therapy, increasing NPs endocytosis and intracellular drug concentration

Microscopic Imaging and Labeling Dataset for the Detection of <i>Pneumocystis jirovecii</i> Using Methenamine Silver Staining Method

Pneumocystis jirovecii pneumonia is one of the diseases that most affects immunocompromised patients today, and under certain circumstances, it can be fatal. On the other hand, more and more automatic tools based on artificial intelligence are required every day to help diagnose diseases and thus optimize the resources of the healthcare system. It is therefore important to develop techniques and mechanisms that enable early diagnosis. One of the most widely used techniques in diagnostic laboratories for the detection of its etiological agent, Pneumocystis jirovecii, is optical microscopy. Therefore, an image dataset of 29 different patients is presented in this work, which can be used to detect whether a patient is positive or negative for this fungi. These images were taken in at least four random positions on the specimen holder. The dataset consists of a total of 137 RGB images. Likewise, it contains realistic, annotated, and high-quality microscope images. In addition, we provide image segmentation and labeling that can also be used in numerous studies based on artificial intelligence implementation. The labeling was also validated by an expert, allowing it to be used as a reference in the training of automatic algorithms with supervised learning methods and thus to develop diagnostic assistance systems. Therefore, the dataset will open new opportunities for researchers working in image segmentation, detection, and classification problems related to Pneumocystis jirovecii pneumonia diagnosis

Chemopreventive Effect on Human Colon Adenocarcinoma Cells of Styrylquinolines: Synthesis, Cytotoxicity, Proapoptotic Effect and Molecular Docking Analysis

Seven styrylquinolines were synthesized in this study. Two of these styrylquinolines are new and were elucidated by spectroscopic analysis. The chemopreventive potential of these compounds was evaluated against SW480 human colon adenocarcinoma cells, its metastatic derivative SW620, and normal cells (HaCaT). According to the results, compounds 3a and 3d showed antiproliferative activity in SW480 and SW620 cells, but their effect seemed to be caused by different mechanisms of action. Compound 3a induced apoptosis independent of ROS production, as evidenced by increased levels of caspase 3, and had an immunomodulatory effect, positively regulating the production of different immunological markers in malignant cell lines. In contrast, compound 3d generated a pro-oxidant response and inhibited the growth of cancer cells, probably by another type of cell death other than apoptosis. Molecular docking studies indicated that the most active compound, 3a, could efficiently bind to the proapoptotic human caspases-3 protein, a result that could provide valuable information on the biochemical mechanism for the in vitro cytotoxic response of this compound in SW620 colon carcinoma cell lines. The obtained results suggest that these compounds have chemopreventive potential against CRC, but more studies should be carried out to elucidate the molecular mechanisms of action of each of them in depth

Resveratrol/Hydrazone Hybrids: Synthesis and Chemopreventive Activity against Colorectal Cancer Cells

A series of resveratrol/hydrazone hybrids were obtained and elucidated by spectroscopic analysis. All compounds were evaluated against colorectal cancer cells (SW480 and Sw620) and nonmalignant cell lines (HaCaT and CHO-K1) to establish the selectivity index. Among the hybrids evaluated, compounds 6e and 7 displayed the highest cytotoxic activity with IC50 values of = 6.5 ± 1.9 µM and 19.0 ± 1.4 µM, respectively, on SW480 cells. In addition, hybrid 7 also exhibited activity on SW620 cells with an IC50 value of 38.41 ± 3.3 µM. Both compounds were even more toxic against these malignant cells in comparison to the nonmalignant ones, as evidenced by higher selectivity indices 48 h after treatment. These compounds displayed better activity and selectivity than parental compounds (PIH and Resveratrol) and the reference drug (5-FU). In addition, it was observed that both compounds caused antiproliferative activity probably exerted by cell cycle arrest at the G2/M or G0/G1 phases, with the formation of cells in the subG0/G1 phase. Furthermore, it was noticed that compound 7 induced mitochondrial depolarization in SW480 cells and positive staining for propidium iodide in both cancer cell lines, suggesting cell membrane damage involving either apoptosis or other processes of death

Evaluation of the Effects of Genistein In Vitro as a Chemopreventive Agent for Colorectal Cancer—Strategy to Improve Its Efficiency When Administered Orally

Colorectal Cancer (CRC) ranks third in terms of incidence and second in terms of mortality and prevalence worldwide. In relation to chemotherapy treatment, the most used drug is 5-fluorouracil (5-FU); however, the use of this drug generates various toxic effects at the systemic level. For this reason, new therapeutic strategies are currently being sought that can be used as neoadjuvant or adjuvant treatments. Recent research has shown that natural compounds, such as genistein, have chemotherapeutic and anticancer effects, but the mechanisms of action of genistein and its molecular targets in human colon cells have not been fully elucidated. The results reported in relation to non-malignant cell lines are also unclear, which does not allow evidence of the selectivity that this compound may have. Therefore, in this work, genistein was evaluated in vitro in both cancer cell lines SW480 and SW620 and in the non-malignant cell line HaCaT. The results obtained show that genistein has selectivity for the SW480 and SW620 cell lines. In addition, it inhibits cell viability and has an antiproliferative effect in a dose-dependent manner. Increased production of reactive oxygen species (ROS) was also found, suggesting an association with the cell death process through various mechanisms. Finally, the encapsulation strategy that was proposed made it possible to demonstrate that bacterial nanocellulose (BNC) is capable of protecting genistein from the acidic conditions of gastric fluid and also allows the release of the compound in the colonic fluid. This would allow genistein to act locally in the mucosa of the colon where the first stages of CRC occur