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1.
J Extracell Vesicles ; 10(5): e12071, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33732416

RESUMO

Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract.


Assuntos
Vesículas Extracelulares/metabolismo , Sistema de Sinalização das MAP Quinases , Leite Humano/citologia , Mucosa Bucal/fisiologia , Adulto , Linhagem Celular , Vesículas Extracelulares/imunologia , Feminino , Humanos , Mucosa Bucal/imunologia , Linfócitos T/imunologia , Receptor 3 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Sci Rep ; 10(1): 3008, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080300

RESUMO

Several proteins and peptides in saliva were shown to stimulate gingival wound repair, but the role of salivary metabolites in this process remains unexplored. In vitro gingival re-epithelialization kinetics were determined using unstimulated saliva samples from healthy individuals collected during an experimental gingivitis study. Elastic net regression with stability selection identified a specific metabolite signature in a training dataset that was associated with the observed re-epithelialization kinetics and enabled its prediction for all saliva samples obtained in the clinical study. This signature encompassed ten metabolites, including plasmalogens, diacylglycerol and amino acid derivatives, which reflect enhanced host-microbe interactions. This association is in agreement with the positive correlation of the metabolite signature with the individual's gingival bleeding index. Remarkably, intra-individual signature-variation over time was associated with elevated risk for gingivitis development. Unravelling how these metabolites stimulate wound repair could provide novel avenues towards therapeutic approaches in patients with impaired wound healing capacity.


Assuntos
Eritritol/uso terapêutico , Gengiva/efeitos dos fármacos , Gengivite/metabolismo , Hemorragia/metabolismo , Metaboloma , Saliva/metabolismo , Adolescente , Adulto , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Bioensaio , Estudos de Casos e Controles , Linhagem Celular , Diglicerídeos/metabolismo , Diglicerídeos/farmacologia , Suscetibilidade a Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Gengiva/metabolismo , Gengiva/microbiologia , Gengiva/patologia , Gengivite/tratamento farmacológico , Gengivite/microbiologia , Gengivite/patologia , Hemorragia/tratamento farmacológico , Hemorragia/microbiologia , Hemorragia/patologia , Interações Hospedeiro-Patógeno , Humanos , Masculino , Pessoa de Meia-Idade , Plasmalogênios/metabolismo , Plasmalogênios/farmacologia , Reepitelização/efeitos dos fármacos , Reepitelização/fisiologia , Saliva/química , Saliva/microbiologia , Índice de Gravidade de Doença , Streptococcus mutans/crescimento & desenvolvimento , Streptococcus mutans/patogenicidade
4.
Front Immunol ; 10: 94, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809221

RESUMO

Human milk oligosaccharides (HMO) and prebiotic oligosaccharides are proposed to confer several health benefits to the infant. They shape the microbiota, have anti-inflammatory properties, and support epithelial barrier functioning. However, in order to select the best oligosaccharides for inclusion in infant formulas, there is a need to increase our understanding of the specific effects of HMO and prebiotics on the host immune system. Therefore, we investigated the effects of the HMO sialyllactose (SL), and galactooligosaccharides (GOS) on epithelial barrier functioning, microbiota composition, and SCFA production. The effect of GOS and SL on epithelial barrier functioning and microbiota composition was investigated using in vitro models. Epithelial barrier function was investigated by transcriptome analysis of fully polarized Caco-2 cells exposed for 6 h to SL or GOS. In addition, epithelial cell growth, alkaline phosphatase production, and re-epithelization was studied. Further, we investigated the effect of SL and GOS on microbiota composition and SCFA production using in vitro fecal batch cultures. Transcriptome analysis showed that SL and GOS both induced pathways that regulate cell cycle control. This gene-expression profile translated to a phenotype of halted proliferation and included the induction of alkaline phosphatase activity, a marker of epithelial cell differentiation. SL and GOS also promoted re-epithelialization in an in vitro epithelial wound repair assay. SL and GOS did show distinct modulation of microbiota composition, promoting the outgrowth of Bacteroides and bifidobacteria, respectively, which resulted in distinct changes in SCFA production profiles. Our results show that SL and GOS can both modulate epithelial barrier function by inducing differentiation and epithelial wound repair, but differentially promote the growth of specific genera in the microbiota, which is associated with differential changes in SCFA profiles.


Assuntos
Bacteroides/crescimento & desenvolvimento , Bifidobacterium/crescimento & desenvolvimento , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal/patologia , Lactose/análogos & derivados , Leite Humano/metabolismo , Oligossacarídeos/metabolismo , Ácidos Siálicos/metabolismo , Junções Íntimas/patologia , Fosfatase Alcalina/metabolismo , Células CACO-2 , Ciclo Celular/genética , Diferenciação Celular , Proliferação de Células , Ácidos Graxos Voláteis , Humanos , Lactente , Lactose/metabolismo , Transcriptoma
5.
Gigascience ; 7(7)2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29961849

RESUMO

Background: In vitro scratch assays have been widely used to study the influence of bioactive substances on the processes of cell migration and proliferation that are involved in re-epithelialization. The development of high-throughput microscopy and image analysis has enabled scratch assays to become compatible with high-throughput research. However, effective processing and in-depth analysis of such high-throughput image datasets are far from trivial and require integration of multiple image processing and data extraction software tools. Findings: We developed and implemented a kinetic re-epithelialization analysis pipeline (KREAP) in Galaxy. The KREAP toolbox incorporates freely available image analysis tools and automatically performs image segmentation and feature extraction of each image series, followed by automatic quantification of cells inside and outside the scratched area over time. The enumeration of infiltrating cells over time is modeled to extract three biologically relevant parameters that describe re-epithelialization kinetics. The output of the tools is organized, displayed, and saved in the Galaxy environment for future reference. Conclusions: The KREAP toolbox in Galaxy provides an open-source, easy-to-use, web-based platform for reproducible image processing and data analysis of high-throughput scratch assays. The KREAP toolbox could assist a broad scientific community in the discovery of compounds that are able to modulate re-epithelialization kinetics.


Assuntos
Biologia Computacional/métodos , Reepitelização , Software , Algoritmos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Processamento de Imagem Assistida por Computador/métodos , Cinética , Aprendizado de Máquina , Reconhecimento Automatizado de Padrão , Linguagens de Programação , Reprodutibilidade dos Testes , Cicatrização
6.
Sci Rep ; 7(1): 11100, 2017 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-28894194

RESUMO

Gingival re-epithelialization represents an essential phase of oral wound healing in which epithelial integrity is re-establish. We developed an automated high-throughput re-epithelialization kinetic model, using the gingival epithelial cell line Ca9-22. The model was employed to screen 39 lactic acid bacteria, predominantly including oral isolates, for their capacity to accelerate gingival re-epithelialization. This screen identified several strains of Streptococcus salivarius that stimulated re-epithelialization. Further analysis revealed that S. salivarius strain MS-oral-D6 significantly promoted re-epithelialization through a secreted proteinaceous compound and subsequent experiments identified a secreted serine protease as the most likely candidate to be involved in re-epithelialization stimulation. The identification of bacteria or their products that stimulate gingival wound repair may inspire novel strategies for the maintenance of oral health.


Assuntos
Gengiva/microbiologia , Gengiva/fisiologia , Reepitelização , Serina Proteases/biossíntese , Streptococcus salivarius/fisiologia , Cicatrização , Citocinas/biossíntese , Humanos , Mediadores da Inflamação/metabolismo , Modelos Biológicos , Boca/microbiologia
7.
ISME J ; 11(5): 1218-1231, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28072421

RESUMO

A dysbiotic state is believed to be a key factor in the onset of oral disease. Although oral diseases have been studied for decades, our understanding of oral health, the boundaries of a healthy oral ecosystem and ecological shift toward dysbiosis is still limited. Here, we present the ecobiological heterogeneity of the salivary ecosystem and relations between the salivary microbiome, salivary metabolome and host-related biochemical salivary parameters in 268 healthy adults after overnight fasting. Gender-specific differences in the microbiome and metabolome were observed and were associated with salivary pH and dietary protein intake. Our analysis grouped the individuals into five microbiome and four metabolome-based clusters that significantly related to biochemical parameters of saliva. Low salivary pH and high lysozyme activity were associated with high proportions of streptococcal phylotypes and increased membrane-lipid degradation products. Samples with high salivary pH displayed increased chitinase activity, higher abundance of Veillonella and Prevotella species and higher levels of amino acid fermentation products, suggesting proteolytic adaptation. An over-specialization toward either a proteolytic or a saccharolytic ecotype may indicate a shift toward a dysbiotic state. Their prognostic value and the degree to which these ecotypes are related to increased disease risk remains to be determined.


Assuntos
Metaboloma , Microbiota , Saliva/metabolismo , Saliva/microbiologia , Adolescente , Adulto , Bactérias/classificação , Bactérias/isolamento & purificação , Estudos Transversais , Disbiose , Ecossistema , Feminino , Humanos , Masculino , Adulto Jovem
8.
PLoS One ; 11(12): e0167970, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27941999

RESUMO

Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.


Assuntos
Fímbrias Bacterianas/genética , Lactococcus lactis/genética , Plasmídeos/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Conjugação Genética , Cobre/farmacologia , DNA Bacteriano , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/ultraestrutura , Família Multigênica , Nisina/farmacologia , Análise de Sequência de DNA , Especificidade da Espécie
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