A Screening Approach for Identifying Gliadin Neutralizing Antibodies on Epithelial Intestinal Caco-2 Cells

Hundsberger, Harald; Koppensteiner, Anita; Hofmann, Elisabeth; Ripper, Doris; Pflüger, Maren; Stadlmann, Valerie; Klein, Christian; Kreiseder, Birgit; Katzlinger, Michael; Eger, Andreas; Forster, Florian; Missbichler, Albert; Wiesner, Christoph

doi:10.1177/2472555217697435

Cited by 7 publications

(6 citation statements)

References 28 publications

(47 reference statements)

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“…First, cell viability was assessed using a lactate dehydrogenase (LDH) assay showing no significant cellular death/damage when cells were treated with 0.064 mg mL −1 of p‐gliadin (Figure 6A) as previously reported. [ 47 ]…”

Section: Resultsmentioning

confidence: 99%

Pepsin Digest of Gliadin Forms Spontaneously Amyloid‐Like Nanostructures Influencing the Expression of Selected Pro‐Inflammatory, Chemoattractant, and Apoptotic Genes in Caco‐2 Cells: Implications for Gluten‐Related Disorders

Herrera

Nicoletti

Gras

et al. 2021

Molecular Nutrition Food Res

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Scope Proteolysis‐resistant gliadin peptides are intensely investigated in biomedical research relates to celiac disease and gluten‐related disorders. Herein, the first integrated supramolecular investigation of pepsin‐digested gliadin peptides (p‐gliadin) is presented in combination with its functional behavior in the Caco‐2 cell line. Methods and Results First, gliadins are degraded by pepsin at pH 3, and the physicochemical properties of p‐gliadin are compared with gliadin. An integrated approach using interfacial, spectroscopic, and microscopic techniques reveals that the p‐gliadin forms spontaneously soluble large supramolecular structures, mainly oligomers and fibrils, capable of binding amyloid‐sensitive dyes. The self‐assembly of p‐gliadin starts at a concentration of 0.40 µg mL−1. Second, the stimulation of Caco‐2 cells with the p‐gliadin supramolecular system is performed, and the mRNA expression levels of a panel of genes are tested. The experiments show that p‐gliadin composed of supramolecular structures triggers significant mRNA up‐regulation (p < 0.05) of pro‐apoptotic biomarkers (ratio Bcl2/Bak‐1), chemokines (CCL2, CCL3, CCL4, CCL5, CXCL8), and the chemokine receptor CXCR3. Conclusions This work demonstrates that p‐gliadin is interfacial active, forming spontaneously amyloid‐type structures that trigger genes in the Caco‐2 cell line involved in recruiting specialized immune cells.

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Section: Resultsmentioning

confidence: 99%

Pepsin Digest of Gliadin Forms Spontaneously Amyloid‐Like Nanostructures Influencing the Expression of Selected Pro‐Inflammatory, Chemoattractant, and Apoptotic Genes in Caco‐2 Cells: Implications for Gluten‐Related Disorders

Herrera

Nicoletti

Gras

et al. 2021

Molecular Nutrition Food Res

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“…More importantly, they highlight the importance of considering the functions of antibodies generated in response to gluten, rather than their simple presence. For example, some anti-tTG antibodies may decrease the enzymatic activity of tTG [40], and the development of neutralizing antibodies against gliadin [41] suggests that some AGAs generated during CeD may have a protective effect. Explicitly defining the role anti-tTG and anti-gliadin antibodies play in GSE pathogenesis in vivo, requires the use of mouse models where these responses can be modelled and their physiological effects quantified.…”

Section: Discussionmentioning

confidence: 99%

Gluten-free diet exposure prohibits pathobiont expansion and gluten sensitive enteropathy in B cell deficient JH-/- mice

et al. 2022

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In humans, celiac disease (CeD) is a T-cell-driven gluten-sensitive enteropathy (GSE) localized to the small bowel (duodenum). The presence of antibodies specific for gluten- and self-antigens are commonly used diagnostic biomarkers of CeD and are considered to play a role in GSE pathogenesis. Previously, we have described an apparent T-cell-mediated GSE in CD19-/- mice, which develop weak and abnormal B cell responses. Here, we expand on this observation and use a mouse model of complete B cell deficiency (JH-/- mice), to show that absence of a humoral immune response also promotes development of a GSE. Furthermore, 16S analysis of microbial communities in the small intestine demonstrates that a gluten-free diet suppresses the expansion of anaerobic bacteria in the small intestine and colonization of the small intestine by a specific pathobiont. Finally, we also observe that SI enteropathy in mice fed a gluten-rich diet is positively correlated with the abundance of several microbial peptidase genes, which supports that bacterial metabolism of gluten may be an important driver of GSE in our model. Collectively, results from our experiments indicate that JH-/- mice will be a useful resource to investigators seeking to empirically delineate the contribution of humoral immunity on GSE pathogenesis, and support the hypothesis that humoral immunity promotes tolerance to gluten.

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“…The advantages of this method are the high reproducibility of wounds and automated real-time measurements. Therefore, this approach can be used in medium- to high-throughput screening experiments [ 39 , 40 , 41 , 42 ]. However, in contrast to the conventional scratch assay, specific equipment, i.e.…”

Section: In Vitro Models For Wound Healingmentioning

confidence: 99%

Human In Vitro Skin Models for Wound Healing and Wound Healing Disorders

et al. 2023

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Skin wound healing is essential to health and survival. Consequently, high amounts of research effort have been put into investigating the cellular and molecular components involved in the wound healing process. The use of animal experiments has contributed greatly to the knowledge of wound healing, skin diseases, and the exploration of treatment options. However, in addition to ethical concerns, anatomical and physiological inter-species differences often influence the translatability of animal-based studies. Human in vitro skin models, which include essential cellular and structural components for wound healing analyses, would improve the translatability of results and reduce animal experiments during the preclinical evaluation of novel therapy approaches. In this review, we summarize in vitro approaches, which are used to study wound healing as well as wound healing-pathologies such as chronic wounds, keloids, and hypertrophic scars in a human setting.

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A Screening Approach for Identifying Gliadin Neutralizing Antibodies on Epithelial Intestinal Caco-2 Cells

Cited by 7 publications

References 28 publications

Pepsin Digest of Gliadin Forms Spontaneously Amyloid‐Like Nanostructures Influencing the Expression of Selected Pro‐Inflammatory, Chemoattractant, and Apoptotic Genes in Caco‐2 Cells: Implications for Gluten‐Related Disorders

Pepsin Digest of Gliadin Forms Spontaneously Amyloid‐Like Nanostructures Influencing the Expression of Selected Pro‐Inflammatory, Chemoattractant, and Apoptotic Genes in Caco‐2 Cells: Implications for Gluten‐Related Disorders

Gluten-free diet exposure prohibits pathobiont expansion and gluten sensitive enteropathy in B cell deficient JH-/- mice

Human In Vitro Skin Models for Wound Healing and Wound Healing Disorders

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