Impacts of foodborne inorganic nanoparticles on the gut microbiota-immune axis: potential consequences for host health.

BACKGROUND: In food toxicology, there is growing interest in studying the impacts of foodborne nanoparticles (NPs, originating from food additives, food supplements or food packaging) on the intestinal microbiome due to the important and complex physiological roles of these microbial communities in host health. Biocidal activities, as described over recent years for most inorganic and metal NPs, could favour chronic changes in the composition and/or metabolic activities of commensal bacteria (namely, intestinal dysbiosis) with consequences on immune functions. Reciprocally, direct interactions of NPs with the immune system (e.g., inflammatory responses, adjuvant or immunosuppressive properties) may in turn have effects on the gut microbiota. Many chronic diseases in humans are associated with alterations along the microbiota-immune system axis, such as inflammatory bowel diseases (IBD) (Crohn's disease and ulcerative colitis), metabolic disorders (e.g., obesity) or colorectal cancer (CRC). This raises the question of whether chronic dietary exposure to inorganic NPs may be viewed as a risk factor facilitating disease onset and/or progression. Deciphering the variety of effects along the microbiota-immune axis may aid the understanding of how daily exposure to inorganic NPs through various foodstuffs may potentially disturb the intricate dialogue between gut commensals and immunity, hence increasing the vulnerability of the host. In animal studies, dose levels and durations of oral treatment are key factors for mimicking exposure conditions to which humans are or may be exposed through the diet on a daily basis, and are needed for hazard identification and risk assessment of foodborne NPs. This review summarizes relevant studies to support the development of predictive toxicological models that account for the gut microbiota-immune axis. CONCLUSIONS: The literature indicates that, in addition to evoking immune dysfunctions in the gut, inorganic NPs exhibit a moderate to extensive impact on intestinal microbiota composition and activity, highlighting a recurrent signature that favours colonization of the intestine by pathobionts at the expense of beneficial bacterial strains, as observed in IBD, CRC and obesity. Considering the long-term exposure via food, the effects of NPs on the gut microbiome should be considered in human health risk assessment, especially when a nanomaterial exhibits antimicrobial properties.

Oral delivery of pancreatitis-associated protein by Lactococcus lactis displays protective effects in dinitro-benzenesulfonic-acid-induced colitis model and is able to modulate the composition of the microbiota.

Antimicrobial peptides secreted by intestinal immune and epithelial cells are important effectors of innate immunity. They play an essential role in the maintenance of intestinal homeostasis by limiting microbial epithelium interactions and preventing unnecessary microbe-driven inflammation. Pancreatitis-associated protein (PAP) belongs to Regenerating islet-derived III proteins family and is a C-type (Ca+2 dependent) lectin. PAP protein plays a protective effect presenting anti-inflammatory properties able to reduce the severity of colitis, preserving gut barrier and epithelial inflammation. Here, we sought to determine whether PAP delivered at intestinal lumen by recombinant Lactococcus lactis strain (LL-PAP) before and after chemically induced colitis is able to reduce the severity in two models of colitis. After construction and characterization of our recombinant strains, we tested their effects in dinitro-benzenesulfonic-acid (DNBS) and Dextran sulfate sodium (DSS) colitis model. After the DNBS challenge, mice treated with LL-PAP presented less severe colitis compared with PBS and LL-empty-treated mice groups. After the DSS challenge, no protective effects of LL-PAP could be detected. We determined that after 5 days administration, LL-PAP increase butyrate producer's bacteria, especially Eubacterium plexicaudatum. Based on our findings, we hypothesize that a treatment with LL-PAP shifts the microbiota preventing the severity of colon inflammation in DNBS colitis model. These protective roles of LL-PAP in DNBS colitis model might be through intestinal microbiota modulation.

Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis.

BACKGROUND: Mucositis is one of the most relevant gastrointestinal inflammatory conditions in humans, generated by the use of chemotherapy drugs, such as 5-fluoracil (5-FU). 5-FU-induced mucositis affects 80% of patients undergoing oncological treatment causing mucosal gut dysfunctions and great discomfort. As current therapy drugs presents limitations in alleviating mucositis symptoms, alternative strategies are being pursued. Recent studies have shown that the antimicrobial pancreatitis-associated protein (PAP) has a protective role in intestinal inflammatory processes. Indeed, it was demonstrated that a recombinant strain of Lactococcus lactis expressing human PAP (LL-PAP) could prevent and improve murine DNBS-induced colitis, an inflammatory bowel disease (IBD) that causes severe inflammation of the colon. Hence, in this study we sought to evaluate the protective effects of LL-PAP on 5-FU-induced experimental mucositis in BALB/c mice as a novel approach to treat the disease. RESULTS: Our results show that non-recombinant L. lactis NZ9000 have antagonistic activity, in vitro, against the enteroinvasive gastrointestinal pathogen L. monocytogenes and confirmed PAP inhibitory effect against Opportunistic E. faecalis. Moreover, L. lactis was able to prevent histological damage, reduce neutrophil and eosinophil infiltration and secretory Immunoglobulin-A in mice injected with 5-FU. Recombinant lactococci carrying antimicrobial PAP did not improve those markers of inflammation, although its expression was associated with villous architecture preservation and increased secretory granules density inside Paneth cells in response to 5-FU inflammation. CONCLUSIONS: We have demonstrated for the first time that L. lactis NZ9000 by itself, is able to prevent 5-FU-induced intestinal inflammation in BALB/c mice. Moreover, PAP delivered by recombinant L. lactis strain showed additional protective effects in mice epithelium, revealing to be a promising strategy to treat intestinal mucositis.

Expression of fibronectin binding protein A (FnBPA) from Staphylococcus aureus at the cell surface of Lactococcus lactis improves its immunomodulatory properties when used as protein delivery vector.

A recombinant strain of Lactococcus lactis displaying a cell-surface anchored fibronectin binding protein A (FnBPA) from Staphylococcus aureus (LL-FnBPA) had been shown to be more efficient in delivering plasmid than its wild-type counterpart both in vitro and in vivo, and have the ability to orientate the immune response toward a Th2 profile in a context of a DNA vaccination. The aim of this work was to test whether this LL-FnBPA strain could shape the immune response after mucosal administration in mice. For this, we used a mouse model of human papilloma virus (HPV)-induced cancer and a L. lactis strain displaying at its cell surface both HPV-16-E7 antigen (LL-E7) and FnBPA (LL-E7+FnBPA). Our results revealed a more efficient systemic Th1 immune response with recombinant LL-E7+FnBPA. Furthermore, mice vaccinated with LL-E7+FnBPA were better protected when challenged with HPV-16-induced tumors. Altogether, the results suggest that FnBPA displays adjuvant properties when used in the context of mucosal delivery using L. lactis as a live vector.

Genotype and expression of the enhanced gree fluorescent protein in LEW-Tg (EGFP) F455.5/Rrrc rats / Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987; Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.

A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP.

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.

Effect of a three-dimensional chitosan porous scaffold on the differentiation of mesenchymal stem cells into chondrocytes.

Cartilage tissue has a poor capacity for self-repair, especially in the case of severe cartilage damage due to trauma or age-related degeneration. Cell-based tissue engineering using scaffolds has provided an option for the repair of cartilage tissue. The present work demonstrates that a three-dimensional (3D) chitosan scaffold increases the efficiency of the adhesion and differentiation of mesenchymal stem cells (MSCs) after the addition of a chondrogenic medium. These culture conditions promoted MSC differentiation into chondrocytes during the first 9 weeks of monolayer or 3D culture in a scaffold composed of chitosan or chitosan/gelatin. The results demonstrated that a chitosan scaffold caused a reduction in alkaline phosphatase production and an increase in the collagen concentration indicating phenotypic changes in the cells. In support of these results, the production of collagen type II by the MSCs cultured in the chitosan scaffold increased after 3 weeks of culture, indicating the beginning of differentiation. However, the addition of gelatin to the chitosan scaffold did not improve on the results obtained with chitosan alone. These results suggest that this 3D chitosan scaffold is a promising candidate for biomaterial implants designed to promote MSC colonization and has applications in regenerative medicine.

Spleen cell proliferation during and after skin myiasis by human bot fly Dermatobia hominis / 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.