Proliferação de células do baço durante e após miíase por Dermatobia hominis

Spleen cells from mice were examined at 5, 10, 15, 20 and 25 days post-infection (dpi) with Dermatobia hominis larva and at 5, 10, 15, 30 and 60 days post-larval emergence (dple). Cell proliferation in vitro assays were carried out with RPMI-1640 medium and larval secretory product (LSP) of D. hominis at 5, 10, 15, 20 and 25 days. When each group of mice was tested against each medium, significance was only seen for 25 dpi, with increasing order: LSP-10 d, -25 d, -5 d, -20 d, -15 d and RPMI. Significant results were also observed when each medium was tested against mice at each dpi or dple. Each dple group vs. each medium produced significant results only for 10 dple, with increasing order: LSP-5 d, -20 d, -25 d, -10 d, -15 d and RPMI. Comparative tests were also carried out between groups to refine certain observations. The LSPs were also analyzed using SDS-PAGE. The results prove that myiasis caused depletion of spleen cells, particularly under the effect of the LSP-10 and -15, but the cells tended to increase up to 60 dple. This in vitro assay may represent the real systemic immune response in the relationship LSP-D. hominis-host.Células do baço de camundongos foram examinadas aos 5, 10, 20 e 25 dias pós-infecção (dpi) com Dermatobia hominis e examinadas aos 5, 10, 15, 30 e 60 dias pós-emergência da larva (dpel). As células foram cultivadas em meio RPMI-1640 contendo, ou não (controle), produtos de secreção das larvas (PSL) de D. hominis com idade de 5, 10, 15, 20 e 25 dias. Em cada grupo com cinco camundongos testados nos meios de cultura, o número de células foi significativo para 25 dpi, com crescente aumento na seguinte ordem: PSL-10 d, -25 d, -5 d, -20 d, -15 d e RPMI. Resultados significantes foram também observados nos testes entre cada meio contendo células tanto de camundongos dpi ou dpel. Em cada dpel grupo versus meio significância foi somente verificada para 10 dpel, na ordem crescente: PSL-5 d, -20 d, -25 d, -10 d, -15 d e RPMI. Testes comparativos foram também realizados entre grupos. O PSL foi analisado sob SDS-PAGE. Os resultados provam que a miíase causou depleção de células do baço, particularmente sob efeito do PSL-10 e -15, mas ocorreu normalidade do número de células aos 60 dpel. Este ensaio in vitro pode representar uma resposta imune sistêmica na relação PSL-D. hominis-hospedeiro

Endothelial Differentiation of Human Stem Cells Seeded onto Electrospun Polyhydroxybutyrate/Polyhydroxybutyrate-Co-Hydroxyvalerate Fiber Mesh

Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This study's aim was to establish a culture of human adipose tissue-derived stem cells (hASCs) to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM). Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF), the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in combination with endothelial-differentiated cells to improve vascularization in engineered bone tissues

Validating intestinal effects of food-grade titanium dioxide using a murine gut organoid model as alternative to in vivo models

International audienceBackground: Nanoparticles (NPs) found in the human diet mainly originate from inorganic food additives, often used quantum satis in common foodstuff, which raises public health concerns due to daily exposure. The whitener and opacifying agent titanium dioxide (TiO2, E171 in EU) is one of the most studied nanomaterial, evoking inflammatory responses and precancerous lesions in the rodent intestine. Investigating the potential hazards of chronic oral exposure to NPs is often time-consuming and requires animal models, specific spaces and skills. However, recent technical advances in stem cells and threedimensional cultures allowed the use of organoids as an alternative model to in vivo experiments. Herein we used murine intestinal organoids to characterize intestinal impacts of food-grade TiO2 in comparison to already reported in vivo data, and to validate organoids as a reliable model for studying the effects of foodborne NPs in the gut.Methods: Three different wild-type C57bl/6 mice were used for small intestine collection. Intestinal crypts were purified, dissociated, and cells were cultured for organoid growth. After 4 passages, organoids were dissociated and seeded as a 2.5D culture, then exposed to 0.1, 1, 10 or 100µg/ml of E171 for 24h. Supernatants were collected, and cytotoxicity assessed by LDH release quantification. Total RNA was extracted from samples and analyzed for cell proliferation and differentiation, genotoxicity, antimicrobial peptides, permeability, oxidative stress, Toll Like Receptors (TLR), NFκB, cytokine and chemokine gene expressions by qPCR. Cell apoptosis was also evaluated by cleaved Caspase-3 quantification using immunofluorescence.Results: Gut organoids exposed to E171 showed a dose-dependent up-regulation of the cell proliferation marker Mki67 together with increased protein expression of cleaved-Caspase-3, suggesting epithelium renewal or restructuring. This occurred in parallel to a decreased expression of the enterocyte differentiation markers Alpi and Krt20 as well as up-regulation of the neuroendocrine marker Chga. Moreover, food-grade E171 decreased gene expression of antimicrobial peptides (Lyz, Reg3b, S100a8) and tight junction proteins (F11r, Tjp1, Ocln, Cldn7, Cldn15), suggesting altered epithelial secretion and permeability. We also showed that the TLR4-NFκB pathway was negatively impacted in a dose-dependent manner, while oxidative stress, cytokine and chemokine gene expressions remained unaltered. Although E171 exposure was not cytotoxic, TiO2 increased expression of gadd45a at low dose (i.e. 1µg/ml), suggesting DNA damage.Conclusions: Taking together, a 24h-exposure of murine intestinal organoids to food-grade TiO2 impacts epithelial barrier integrity (cell proliferation and differentiation, gut permeability, genotoxic effect) and antimicrobial defenses as reported in vivo in rodent models, hence validating the use of intestinal organoids for toxicological studies of foodborne NPs

Protein expression of hASCs during endothelial differentiation.

<p>Confocal images of the expression of the VE-Cadherin (A) and the vWF factor (B) after 21 days. A.1 and B.1 - hASCs cultured with the basal medium on the TCPS coverslips, A.2 and B.2 - hASCs cultured with the endothelial differentiation medium on the TCPS coverslips, A.3 and B.3 - hASCs cultured with the basal medium on the electrospun PHB/PHB-HV fiber mesh, A.4 and B.4 - hASCs cultured with the endothelial differentiation medium on the electrospun PHB/PHB-HV fiber mesh. Scale bar 20 µm. The images, A.2, A.4, B.2 and B.4 represent the overlay of bright-field and confocal images for visualization of the fiber mesh. (C) Fluorescence intensity of the expression of VE-Cadherin and the vWF factor in cells differentiated on TCPS coverslip and electrospun PHB/PHB-HV fiber mesh. The results are expressed as the means ± SD, (*) significant difference for p<0,05. (a.u): arbitrary units.</p

RT-PCR analysis of VEGFR2 mRNA expression during endothelial differentiation on the electrospun PHB/PHB-HV fiber mesh and TCPS.

<p>Total RNA was extracted from cells cultured on basal medium and endothelial differentiation medium for analysis of VEGFR2 mRNA expression. The cells were cultivated up to 21 days.</p

Proliferation and viability of hASCs cultured on TCPS and electrospun PHB/PHB-HV fiber mesh.

<p>(A) MTT proliferation assays performed 7, 14 and 21 days after the cell seeding and cultured in two specific medium: the basal medium and the endothelial differentiation medium. The results are expressed as the means ± SD, (*) indicating a significant difference with p<0.05, (**) p<0,01, (***) p<0,001; (B) cell viability after 21 days of cell culture with the basal medium and (C) the endothelial differentiation medium on the electrospun PHB/PHB-HV fiber mesh, as analyzed by Calcein-AM staining.</p

Flow cytometry analysis of hASCs.

<p>The expression pattern of specific antigens on the surface of the hASCs is depicted with representative histograms and the expression of each marker. The cell population expressed CD29, CD44, CD73 and HLA-ABC, and did not express CD34, CD45 and HLA-DR.</p