RESUMEN
The universal presence of micro-nanoplastics (MNPLs) and its relative unknown effects on human health is a concern demanding reliable data to evaluate their safety. As ingestion is one of the main exposure routes for humans, we have assessed their hazard using two in vitro models that simulate the human intestinal barrier and its associated lymphoid system. Two different coculture models (differentiated Caco-2/HT29 intestinal cells and Caco-2/HT29 + Raji-B cells) were exposed to polystyrene nanoparticles (PSNPs) for 24 h. Endpoints such as viability, membrane integrity, NPS localization and translocation, ROS induction, and genotoxic damage were evaluated to have a comprehensive view of their potentially harmful effects. No significant cytotoxic effects were observed in any of the analyzed systems. In addition, no adverse effects were detected in the integrity or in the permeability of the barrier model. Nevertheless, confocal microscopy analysis showed that MNPLs were highly uptaken by both of the barrier model systems, and that translocation across the membrane occurred. Thus, MNPLs were detected into Raji-B cells, placed in the basolateral compartment of the insert. The internalization followed a dose-dependent pattern, as assessed by flow cytometry. Nonetheless, no genotoxic or oxidative DNA damage induction was detected in either case. Finally, no variations in the transcription of oxidative and stress genes could be detected in any of the in vitro barrier models. Our results show that MNPLs can enter and cross the epithelial barrier of the digestive system, as demonstrated when Raji-B cells were included in the model, but without exerting apparent hazardous effects.
Asunto(s)
Intestinos , Microplásticos/toxicidad , Poliestirenos/toxicidad , Células CACO-2 , Diferenciación Celular , Daño del ADN , Células HT29 , Humanos , Microscopía Confocal , Nanopartículas , PermeabilidadRESUMEN
The use of in vitro barrier models is gaining relevance as an alternative to animal studies in risk assessment, pharmacokinetic, and toxicological studies in general. These models permit an easier evaluation of the underlying mechanisms taking place at the molecular and cellular levels on the barrier site. Here, we report several methodological modifications of the three-dimensional in vitro intestinal epithelial model Caco-2/HT29/Raji-B for its successful application in the Nanotoxicology field. In addition, new insights in the study of specific molecular markers and new confocal microscope approaches have also been incorporated. Due to the multiple variables and parameters playing a part when the model's complexity is increased, we have monitored the barrier's formation and cell differentiation over time. Finally, the practical usability of the proposed model was tested by evaluating the action of the food additives titanium dioxide and silica dioxide nanoparticles (TiO2NPs and SiO2NPs). The NPs-associated effects were evaluated by confocal microscopy. We have demonstrated the essential role of the mucus layer in the decrease of cellular uptake, avoiding potential NPs-cell nuclei interactions.
Asunto(s)
Técnicas de Cocultivo , Mucosa Intestinal/efectos de los fármacos , Nanopartículas/toxicidad , Dióxido de Silicio/toxicidad , Titanio/toxicidad , Células CACO-2 , Diferenciación Celular , Línea Celular , Aditivos Alimentarios/toxicidad , Industria de Alimentos , Expresión Génica , Células HT29 , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Microscopía ConfocalRESUMEN
Salmonella enterica serovar Typhi causes the systemic disease typhoid fever. After ingestion, it adheres to and invades the host epithelium while evading the host innate immune response, causing little if any inflammation. Conversely, Salmonella enterica serovar Typhimurium causes gastroenteritis in humans and thrives in the inflamed gut. Upon entering the host, S Typhimurium preferentially colonizes Peyer's patches, a lymphoid organ in which microfold cells (M cells) overlay an arrangement of B cells, T cells, and antigen-presenting cells. Both serovars can adhere to and invade M cells and enterocytes, and it has been assumed that S Typhi also preferentially targets M cells. In this study, we present data supporting the alternative hypothesis that S Typhi preferentially targets enterocytes. Using a tissue culture M cell model, we examined S Typhi strains with a deletion in the stg fimbriae. The stg deletion resulted in increased adherence to M cells and, as expected, decreased adherence to Caco-2 cells. Adherence to M cells could be further enhanced by introduction of the long polar fimbriae (Lpf), which facilitate adherence of S Typhimurium to M cells. Deletion of stg and/or introduction of lpf enhanced M cell invasion as well, leading to significant increases in secretion of interleukin 8. These results suggest that S Typhi may preferentially target enterocytes in vivo.
Asunto(s)
Adhesión Bacteriana , Enterocitos/microbiología , Fimbrias Bacterianas/metabolismo , Salmonella typhi/fisiología , Células CACO-2 , HumanosRESUMEN
HLA-DR is the most commonly expressed and likely the most medically important human MHC class II, antigen presenting protein. In a normal immune response, HLA-DR binds to antigenic peptide and the HLA-DR/peptide complex binds to a T-cell receptor, thus contributing to T-cell activation and stimulation of an immune response against the antigen. When foreign antigen is not present, HLA-DR binds endogenous peptide which, under normal conditions does not stimulate an immune response. In most cases, the human peptide is CLIP, but a certain percentage of HLA-DR molecules will be present at the cell surface with other human peptides. We have recently shown that cell surface, CLIP/HLA-DR ratios are a measure of peptide heterogeneity, and in particular, changes in CLIP/HLA-DR ratios represent changes in the occupancy of HLA-DR by other, endogenous peptides. For example, treatment of cells with the HDAC inhibitor, Entinostat, leads to an upregulation of Cathepsin L1 and replacement of Cathepsin L1 senstitive peptides with HLA-DR binding, Cathepsin L1 resistant peptides, an alteration that can be at least partially assessed via assessment of CLIP/HLA-DR cell surface ratios. Here we assay for CLIP/HLA-DR ratios following treatment of immortalized B-cells with a variety of common drugs, almost all of which indicate significant changes in the CLIP/HLA-DR ratios. Furthermore, the CLIP/HLA-DR ratio changes parallel the impact of the drug panoply on cell viability, suggesting that alterations in the HLA-DR peptidome are governed by a variety of mechanisms, rather than exclusively dependent on a dedicated peptide loading process. These results raise questions about how FDA approved drugs may affect the immune response, and whether any of these drugs could be useful as vaccine adjuvants?
Asunto(s)
Presentación de Antígeno/efectos de los fármacos , Antígenos de Diferenciación de Linfocitos B/análisis , Linfocitos B/química , Linfocitos B/efectos de los fármacos , Antígenos HLA-DR/análisis , Antígenos de Histocompatibilidad Clase II/análisis , HumanosRESUMEN
Caco-2 based cell models have been the gold standard in vitro method to study intestinal drug permeability, despite the absence of many important features with major influence in the drug absorption mechanism. In the present work, a triple co-culture comprising Caco-2, HT29-MTX and Raji B cells was established to mimic in a closely way the human intestinal epithelium, presenting the main components in the process of drug absorption, namely the absorptive cells that resemble enterocytes, mucus producers cells and cells able to induce M-cell phenotype on Caco-2 cells. All the three cell lines maintained their function when cultured together with each other being, thus, an asset to new orally administrated drugs development. The seeding ratio of 90:10 between Caco-2 and HT29-MTX showed to be the best to achieve physiological proportions after cells maturation and differentiation in culture. The formation of M-cells phenotype from enterocytes was identified for the first time in a co-culture system comprising Caco-2 and HT29-MTX cells through immunocytochemical techniques. Thus, the triple co-culture model presented in the herein work is a good and reliable alternative to the in vitro methods already existents for the study of drugs permeability.