The Immunoreactive Protein was Produced During Absorption of Glycinin or its Hydrolysate in IPEC-J2

2017 ◽  
Vol 13 (9) ◽  
Author(s):  
Yuan Zhao ◽  
Shiyao Zhang ◽  
Gaowa Naren ◽  
Guixin Qin
Keyword(s):  
Mass Spectrometry ◽  
Epithelial Cells ◽  
Immunosorbent Assay ◽  
Intestinal Epithelial Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intestinal Epithelial ◽  
Intracellular Accumulation ◽  
Immunoreactive Protein ◽  
Gut Epithelium

AbstractThe allergens absorbed in immunoreactive form by the gut epithelium might induce the occurrence of allergy. The purpose of this study was to investigate the absorption and intracellular accumulation of the intact or hydrolyzed glycinin in the porcine intestinal epithelial cells (IPEC-J2). The IPEC-J2 cells were incubated by 0, 0.25, 0.5 and 1.0 mg/mL glycinin or its hydrolysate for 2, 4, 8 or 12 h. The amounts of immunoreactive glycinin were measured by enzyme-linked immunosorbent assay. The intact and hydrolyzed glycinin fragments of epithelial absorption were identified by immunoblotting and mass spectrometry (MS). We found that glycinin or its hydrolysate is expensively absorbed with the increase of dose and time. The 35 kD or 22 kD protein with glycinin-specific epitopes was detected in the intracellular extracts and basolateral solutions. The results indicate that the glycinin or its hydrolysate could be absorbed; meanwhile, the 35kD or 22kD protein was correspondingly produced during absorption.

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.

Differential proteomic analysis of HT29 Cl.16E and intestinal epithelial cells by LC ESI/QTOF mass spectrometry

2009 ◽  
Vol 72 (5) ◽  
pp. 865-873 ◽  
Author(s):  
Paolo Nanni ◽  
Laura Mezzanotte ◽  
Giulia Roda ◽  
Alessandra Caponi ◽  
Fredrik Levander ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Epithelial Cells ◽  
Proteomic Analysis ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial

scholarly journals JunD enhances miR-29b levels transcriptionally and posttranscriptionally to inhibit proliferation of intestinal epithelial cells

2015 ◽  
Vol 308 (10) ◽  
pp. C813-C824 ◽  
Author(s):  
Tongtong Zou ◽  
Jaladanki N. Rao ◽  
Lan Liu ◽  
Lan Xiao ◽  
Hee Kyoung Chung ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcription Factor ◽  
Epithelial Cells ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Intestinal Epithelial Cells ◽  
Inhibitory Effect ◽  
Microrna Expression ◽  
Intestinal Epithelial ◽  
Gut Epithelium

Through its actions as component of the activating protein-1 (AP-1) transcription factor, JunD potently represses cell proliferation. Here we report a novel function of JunD in the regulation of microRNA expression in intestinal epithelial cells (IECs). Ectopically expressed JunD specifically increased the expression of primary and mature forms of miR-29b, whereas JunD silencing inhibited miR-29b expression. JunD directly interacted with the miR-29b1 promoter via AP-1-binding sites, whereas mutation of AP-1 sites from the miR-29b1 promoter prevented JunD-mediated transcriptional activation of the miR-29b1 gene. JunD also enhanced formation of the Drosha microprocessor complex, thus further promoting miR-29b biogenesis. Cellular polyamines were found to regulate miR-29b expression by altering JunD abundance, since the increase in miR-29b expression levels in polyamine-deficient cells was abolished by JunD silencing. In addition, miR-29b silencing prevented JunD-induced repression of IEC proliferation. Our findings indicate that JunD activates miR-29b by enhancing its transcription and processing, which contribute to the inhibitory effect of JunD on IEC growth and maintenance of gut epithelium homeostasis.

scholarly journals Geniposide, a Component of Gardenia Jasminoides Ellis, Inhibits NF-ĸb to Attenuate LPS-Induced Injury in Intestinal Epithelial Cells

2021 ◽  
Author(s):  
Qiuju Wang ◽  
Xinyue Qiao ◽  
Mengzu Wang ◽  
Junfeng Jia ◽  
and Yizhe Cui
Keyword(s):  
Epithelial Cells ◽  
Inflammatory Responses ◽  
Intestinal Epithelial Cells ◽  
Interleukin 1 ◽  
Inflammatory Factors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intestinal Epithelial ◽  
Migration Ability ◽  
Gardenia Jasminoides ◽  
Gardenia Jasminoides Ellis

The nuclear factor-ĸB (NF-ĸB) transcriptional system is a major effector pathway involved in inflammatory responses. Previous studies found that a Gardenia decoction (GD) inhibited the expression of NF-&kappa;B in a lipopolysaccharide (LPS)-stimulated mouse intestinal injury model. Herein, we hypothesized that geniposide (GE), a component of Gardenia jasminoides Ellis, also exerts anti-inflammatory effects and inhibits NF-ĸB activity in LPS-induced intestinal epithelial cells (IEC-6). IEC-6 cells were stimulated with LPS, following which the effects of GE on NF-ĸB signaling in the IEC-6 cells were examined by western blotting to detect IĸB phosphorylation/degradation. The expression of NF-&kappa;B was determined by immunofluorescence assay (IFA). Enzyme-linked immunosorbent assay (ELISA) was used to detect the inhibitory effect of GE on the release of tumor necrosis factor-&alpha; (TNF-&alpha;), interleukin-6 (IL-6) and interleukin-1&beta; (IL-1&beta;) activated by LPS in IEC-6 cells. In addition, the migration ability of IEC-6 cells was observed by the scratch method. These results showed that GE dose-dependently downregulated levels of the proinflammatory cytokines TNF-&alpha;, IL-6 and IL-1&beta; that had been upregulated by LPS and suppressed the phosphorylation of IĸB and NF-ĸB induced by LPS. Our findings indicated that GE could reduce LPS-induced NF-ĸB signaling and proinflammatory expression in IEC-6 cells and significantly enhance the migration of IEC-6 cells. Moreover, GE inhibited the expression of NF-&kappa;B, nuclear transfer, and transcriptional activity in IEC-6 cells. GE could block the synthesis of inflammatory factors of IEC-6 cells by inhibiting activation of the IĸB/NF-&kappa;B signaling pathway induced by LPS.

scholarly journals Serum levels of NLRC4 and MCP-2/CCL8 in patients with active Crohn’s disease

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260034
Author(s):  
Kader Irak ◽  
Mehmet Bayram ◽  
Sami Cifci ◽  
Gulsen Sener
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Serum Levels ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intestinal Epithelial ◽  
Intestinal Homeostasis ◽  
Median Serum ◽  
Circulating Levels

Crohn’s disease (CD) is characterized by malfunction of immune-regulatory mechanisms with disturbed intestinal mucosal homeostasis and increased activation of mucosal immune cells, leading to abnormal secretion of numerous pro- and anti-inflammatory mediators. MCP2/CCL8 is produced by intestinal epithelial cells and macrophages, and is a critical regulator of mucosal inflammation. NLRC4 is expressed in phagocytes and intestinal epithelial cells and is involved in intestinal homeostasis and host defense. However, no study to date has assessed the circulating levels of NLRC4 and MCP2/CCL8 in patients with CD. The study was aimed to investigate the serum levels of MCP2/CCL8 and NLRC4 in patients with active CD. Sixty-nine patients with active CD and 60 healthy participants were included in the study. Serum levels of NLRC4 and MCP2/CCL8 were determined using an enzyme-linked immunosorbent assay. The median serum NLRC4 levels were lower in the patient group than in the controls (71.02 (range, 46.59–85.51) pg/mL vs. 99.43 (range 83.52–137.79) pg/mL) (P < 0.001). The median serum levels of MCP2/CCL8 were decreased in patients with CD (28.68 (range, 20.16–46.0) pg/mL) compared with the controls (59.96 (range, 40.22–105.59) pg/mL) (P < 0.001). Cut-off points of NLRC4 (<81 pg/mL) and MCP2/CCL8 (<40 pg/mL) showed high sensitivity and specificity for identifying active CD. In conclusion, this is the first study to examine circulating levels of MCP2/CCL8 and NLRC4 in patients with active CD. Our results suggest that serum NLRC4 and MCP2/CCL8 levels may be involved in the pathogenesis of CD and may have a protective effect on intestinal homeostasis and inflammation. Serum levels of MCP2/CCL8 and NLRC4 could be used as a diagnostic tool and therapeutic target for CD.

scholarly journals Intestinal Epithelial Cells Express Immunomodulatory ISG15 During Active Ulcerative Colitis and Crohn’s Disease

2020 ◽  
Vol 14 (7) ◽  
pp. 920-934 ◽  
Author(s):  
Ann Elisabet Østvik ◽  
Tarjei Dahl Svendsen ◽  
Atle van Beelen Granlund ◽  
Berit Doseth ◽  
Helene Kolstad Skovdahl ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
In Situ Hybridisation ◽  
Gene Signature ◽  
Enzyme Linked Immunosorbent Assay ◽  
Special Focus ◽  
Type I ◽  
Intestinal Epithelial ◽  
Western Blots ◽  
Gene And Protein Expression

Abstract Background and Aims Intestinal epithelial cells [IECs] secrete cytokines that recruit immune cells to the mucosa and regulate immune responses that drive inflammation in inflammatory bowel disease [IBD]. However, experiments in patient-derived IEC models are still scarce. Here, we aimed to investigate how innate immunity and IEC-specific pattern recognition receptor [PRR] signalling can be involved in an enhanced type I interferon [IFN] gene signature observed in colon epithelium of patients with active IBD, with a special focus on secreted ubiquitin-like protein ISG15. Methods Gene and protein expression in whole mucosa biopsies and in microdissected human colonic epithelial lining, in HT29 human intestinal epithelial cells and primary 3D colonoids treated with PRR-ligands and cytokines, were detected by transcriptomics, in situ hybridisation, immunohistochemistry, western blots, and enzyme-linked immunosorbent assay [ELISA]. Effects of IEC-secreted cytokines were examined in human peripheral blood mononuclear cells [PBMCs] by multiplex chemokine profiling and ELISA. Results The type I IFN gene signature in human mucosal biopsies was mimicked in Toll-like receptor TLR3 and to some extent tumour necrosis factor [TNF]-treated human IECs. In intestinal biopsies, ISG15 expression correlated with expression of the newly identified receptor for extracellular ISG15, LFA-1 integrin. ISG15 was expressed and secreted from HT29 cells and primary 3D colonoids through both JAK1-pSTAT-IRF9-dependent and independent pathways. In experiments using PBMCs, we show that ISG15 releases IBD-relevant proinflammatory cytokines such as CXCL1, CXCL5, CXCL8, CCL20, IL1, IL6, TNF, and IFNγ. Conclusions ISG15 is secreted from primary IECs upon extracellular stimulation, and mucosal ISG15 emerges as an intriguing candidate for immunotherapy in IBD.

scholarly journals Dysregulation of Intestinal Microbiota Elicited by Food Allergy Induces IgA-Mediated Oral Dysbiosis

2019 ◽  
Vol 88 (1) ◽  
Author(s):  
Shohei Matsui ◽  
Hideo Kataoka ◽  
Jun-Ichi Tanaka ◽  
Mariko Kikuchi ◽  
Haruka Fukamachi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Food Allergy ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Oral Bacteria ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fecal Bacteria ◽  
Intestinal Epithelial ◽  
Salivary Iga ◽  
Colon 26

ABSTRACT Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown. In this study, we analyzed effects of gut and oral dysbiosis on development of food allergy. A murine model of food allergy was established via ovalbumin (OVA) injection in BALB/c mice. Viable fecal bacteria were identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). il33 expression in colon-26 mouse colon cells stimulated by isolated fecal bacteria was quantified by real-time PCR. Intestinal T cells from the mice were analyzed by flow cytometry. Salivary IgA levels were quantified by enzyme-linked immunosorbent assay (ELISA), and IgA-bound oral bacteria were detected by flow cytometry. Among fecal bacteria, the abundance of Citrobacter sp. increased in the feces of allergic mice and induced il33 expression in colon-26 cells. Orally administered Citrobacter koseri JCM1658 exacerbated systemic allergic symptoms and reduced intestinal Th17 cells. Salivary IgA and IgA-bound oral bacteria increased in the allergic mice. Based on the results described above, food allergy induced both gut and oral dysbiosis. Citrobacter sp. aggravated allergy symptoms by inducing IL-33 release from intestinal epithelial cells.

scholarly journals Geniposide, a component of Gardenia jasminoides Ellis, inhibits NF-ĸB to attenuate LPS-induced injury in intestinal epithelial cells

2020 ◽  
Author(s):  
Yizhe Cui ◽  
Xinyue Qiao ◽  
Qiuju Wang ◽  
Rui Wu
Keyword(s):  
Epithelial Cells ◽  
Inflammatory Responses ◽  
Intestinal Epithelial Cells ◽  
Inflammatory Factors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intestinal Epithelial ◽  
Migration Ability ◽  
Gardenia Jasminoides ◽  
Tnf Α ◽  
Gardenia Jasminoides Ellis

Abstract Background: The nuclear factor-ĸB (NF-ĸB) transcriptional system is a major effector pathway involved in inflammatory responses. Previous studies found that a Gardenia decoction (GD) inhibited the expression of NF-κB in a lipopolysaccharide (LPS)-stimulated mouse intestinal injury model. Herein, we hypothesized that geniposide (GE), a component of Gardenia jasminoides Ellis, also exerts anti-inflammatory effects and inhibits NF-ĸB activity in LPS-induced intestinal epithelial cells (IEC-6). IEC-6 cells were stimulated with LPS, following which the effects of GE on NF-ĸB signaling in the IEC-6 cells were examined by western blotting to detect IĸB phosphorylation/degradation. The expression of NF-κB was determined by immunofluorescence assay (IFA). Enzyme-linked immunosorbent assay (ELISA) was used to detect the inhibitory effect of GE on the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) activated by LPS in IEC-6 cells. In addition, the migration ability of IEC-6 cells was observed by the scratch method. Results: These results showed that GE dose-dependently downregulated levels of the proinflammatory cytokines TNF-α, IL-6 and IL-1β that had been upregulated by LPS and suppressed the phosphorylation of IĸB and NF-ĸB induced by LPS. Our findings indicated that GE could reduce LPS-induced NF-ĸB signaling and proinflammatory expression in IEC-6 cells and significantly enhance the migration of IEC-6 cells. Moreover, GE inhibited the expression of NF-κB, nuclear transfer, and transcriptional activity in IEC-6 cells. Conclusion: GE could block the synthesis of inflammatory factors of IEC-6 cells by inhibiting activation of the IĸB/NF-κB signaling pathway induced by LPS.

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