scholarly journals Specific and Non-Invasive Fluorescent Labelling of Extracellular Vesicles for Evaluation of Intracellular Processing by Intestinal Epithelial Cells

Biomedicines ◽  
2020 ◽  
Vol 8 (7) ◽  
pp. 211
Author(s):  
Maria S. Hansen ◽  
Ida S. E. Gadegaard ◽  
Eva C. Arnspang ◽  
Kristine Blans ◽  
Lene N. Nejsum ◽  
...  
Keyword(s):  
Immune System ◽  
Epithelial Cells ◽  
Fluorescence Microscopy ◽  
Cell Line ◽  
Extracellular Vesicles ◽  
Intestinal Epithelial Cells ◽  
Time Dependent ◽  
Intestinal Epithelial ◽  
Fluorescent Labelling ◽  
Non Invasive

The presence of extracellular vesicles (EVs) in milk has gained interest due to their capacity to modulate the infant’s intestinal and immune system. Studies suggest that milk EVs are enriched in immune-modulating proteins and miRNA, highlighting their possible health benefits to infants. To assess uptake of milk EVs by intestinal epithelial cells, a method was developed using labelling of isolated EVs with fluorophore-conjugated lactadherin. Lactadherin is a generic and validated EV marker, which enables an effective labelling of phosphatidylserine (PS) exposing EVs. Labelled EVs could effectively be used to describe a dose- and time-dependent uptake into the intestinal epithelial Caco-2 cell line. Additionally, fluorescence microscopy was employed to show that EVs colocalize with endosomal markers and lysosomes, indicating that EVs are taken up via general endocytotic mechanisms. Collectively, a method to specifically label isolated EVs is presented and employed to study the uptake of milk EVs by intestinal epithelial cells.

Studying Permeability in a Commonly Used Epithelial Cell Line: T84 Intestinal Epithelial Cells

2011 ◽  
pp. 115-137 ◽  
Author(s):  
Rino P. Donato ◽  
Adaweyah El-Merhibi ◽  
Batjargal Gundsambuu ◽  
Kai Yan Mak ◽  
Emma R. Formosa ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Intestinal Epithelial Cells ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial

Fructooligosaccharide Inhibits the Absorption of β-conglycinin (A Major Soybean Allergen) in IPEC-J2

2018 ◽  
Vol 15 (1-2) ◽  
Author(s):  
Yuan Zhao ◽  
Shiyao Zhang ◽  
Xiaodong Zhang ◽  
Li Pan ◽  
Nan Bao ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Enzymatic Hydrolysis ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Time Dependent ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Intracellular Accumulation ◽  
Novel Therapeutic ◽  
Soybean Allergen

AbstractDissecting the inhibited variation of allergen absorption could contribute to the development of novel therapeutic or preventive treatments for food/feed allergies. This study investigated the effects of fructooligosaccharide (FOS) on the absorption, intracellular accumulation of intact or hydrolysed β-conglycinin in porcine intestinal epithelial cells (IPEC-J2). As demonstrated by ELISA and immunoblotting, β-conglycinin was absorbed in a dose- and time-dependent manner (p < 0.05). Actually, β-conglycinin was easily transported and absorbed after enzymatic hydrolysis. Three peptides (52 kDa, 30 kDa and 25 kDa) were produced during transcellular absorption of intact or hydrolysed β-conglycinin. FOS inhibited the absorption of β-conglycinin, especially the 52 and 30 kDa peptides. The immunoreactive peptides derived from the 52, 35 or 22 kDa peptides were the substrings of the known epitopes determined by mass spectrometry and bioinformatic analyses. These results indicate that FOS can efficiently inhibit the absorption of 52 and 30 kDa peptides derived from β-conglycinin.

scholarly journals The Hippo Pathway Effector YAP1 Regulates Intestinal Epithelial Cell Differentiation

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1895 ◽  
Author(s):  
Sepideh Fallah ◽  
Jean-François Beaulieu
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Epithelial Cells ◽  
Cell Line ◽  
Intestinal Epithelial Cells ◽  
Hippo Pathway ◽  
Intestinal Cell ◽  
Intestinal Epithelial ◽  
The Hippo Pathway

The human intestine is covered by epithelium, which is continuously replaced by new cells provided by stem cells located at the bottom of the glands. The maintenance of intestinal stem cells is supported by a niche which is composed of several signaling proteins including the Hippo pathway effectors YAP1/TAZ. The role of YAP1/TAZ in cell proliferation and regeneration is well documented but their involvement on the differentiation of intestinal epithelial cells is unclear. In the present study, the role of YAP1/TAZ on the differentiation of intestinal epithelial cells was investigated using the HT29 cell line, the only multipotent intestinal cell line available, with a combination of knockdown approaches. The expression of intestinal differentiation cell markers was tested by qPCR, Western blot, indirect immunofluorescence and electron microscopy analyses. The results show that TAZ is not expressed while the abolition of YAP1 expression led to a sharp increase in goblet and absorptive cell differentiation and reduction of some stem cell markers. Further studies using double knockdown experiments revealed that most of these effects resulting from YAP1 abolition are mediated by CDX2, a key intestinal cell transcription factor. In conclusion, our results indicate that YAP1/TAZ negatively regulate the differentiation of intestinal epithelial cells through the inhibition of CDX2 expression.

scholarly journals The function of macromolecular complex of CFTR-NHERF2-LPA2 in inflammatory responses of intestinal epithelial cells

2017 ◽  
Author(s):  
Shanshan Kong ◽  
Weiqiang Zhang
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Human Cell ◽  
Inflammatory Responses ◽  
Intestinal Epithelial Cells ◽  
Human Cell Line ◽  
Macromolecular Complex ◽  
Apical Surface ◽  
Intestinal Epithelial

AbstractCFTR is a cAMP-regulated chloride channel located in the apical surface of intestinal epithelial cells; where it forms a macromolecular complex with NHERF2 and LPA2. CFTR has been shown to play a role in the pathogenies of several types of secretory diarrheas. Inflammatory bowel disease (IBD) is a chronic condition of intestine characterized by severe inflammation and mucosal destruction, genetic analysis has shown that LPA contribute to IBD and patients of cystic fibrosis also display the phenotype of diarrhea. The purpose of this study is to investigate if this complex plays a role in the inflammatory responses of intestinal epithelium.We then explored the role of this complex in maintaining the integrity of tight junction and inflammatory responses in these cells. In vitro assays show that inhibiting CFTR or LPA2 in the intestinal epithelial cell could disrupt the epithelial cell junction, and reduce the TER of intestinal epithelial cells in both mouse and human cell line. EƯSA assay show that intriguing LPA2 through LPS or LPA can increase the secretion of IL-8, while inhibiting or SiRNA knockdown of LPA2 can decrease the secretion of IL-8 in mouse or human intestinal epithelial cells. The CFTR inhibitor can reduce the IL-8 secretion in both mouse and human cell line, the deletion of CFTR in mouse intestine does not affect the IL-8 level, but the knockdown of CFTR in human cell line reduced the IL-8 protein level. The deletion of CFTR in human also reduced the IL-8 mRNA level. This indicates the CFTR-LPA complex is necessary for the expression of IL-8.

Faecal microRNAs: indicators of imbalance at the host-microbe interface?

2018 ◽  
Vol 9 (2) ◽  
pp. 175-183 ◽  
Author(s):  
G.M. Moloney ◽  
M.F. Viola ◽  
A.E. Hoban ◽  
T.G. Dinan ◽  
J.F. Cryan
Keyword(s):  
Epithelial Cells ◽  
Gut Microbiota ◽  
Antibiotic Treatment ◽  
Mirna Expression ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Sprague Dawley ◽  
Faecal Pellets ◽  
Non Invasive ◽  
Bodily Fluids

The enteric microbiota is characterised by a balance and composition that is unique to the host. It is important to understand the mechanisms through which the host can maintain the composition of the gut microbiota. MicroRNAs (miRNA) are implicated in intercellular communication and have been isolated from bodily fluids including stool. Recent findings suggest that miRNA produced by the host’s intestinal epithelial cells (IECs) participate in shaping the microbiota. To investigate whether miRNA expression was influenced by the gut microbiota we measured the expression of miRNAs expressed by intestinal epithelial cells in faeces. Specifically, we measured miRNA expression in faeces from germ-free (GF) and conventional mice and similarly in a rat model of antibiotic-mediated depletion of the gut microbiota control rats. In adult male GF and conventional mice and adult Sprague Dawley (SD) rats were treated with a combination of antibiotics for 8 weeks; total RNA was extracted from faecal pellets taken at week 0, 2, 4, 6 week 8 and the expression of let-7b-3p, miR-141-3p, miR-200a-3p and miR-1224-5p (miRNAs known to be expressed in IECs) were measured relative to U6 at each time point using qRT-PCR. In GF animals the expression of let-7b, miR-141 and miR-200a in faeces was lower compared to conventional mice. Following antibiotic-mediated depletion of gut microbiota, rats showed two divergent profiles of miRNA expression. Following two weeks of antibiotic treatment, the expression of let-7b and miR-1224 dropped significantly and remained low for the remainder of the study. The expression of miR-200a and miR-141 was significantly higher at week 2 than before antibiotic treatment commenced. Subsequently, the expression of miR-200a and miR-141 decreased at week 4 and continued to decrease at week 6. This data demonstrates that miRNAs can be used as an independent, non-invasive marker of microbial fluctuations along with gut pathology in the intestine.

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