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1.
Mol Nutr Food Res ; 68(15): e2400010, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38958100

RESUMO

SCOPE: Celiac disease (CD) is an allergic intestinal disease caused mainly by gliadin in wheat, which is widespread in the population and currently lacks effective treatment. α-Gliadin peptides cause cellular damage by substantially increasing cellular reactive oxygen species (ROS) levels. METHODS AND RESULTS: This study investigates the protective effect of 11 pea-derived peptides (PPs) on ɑ-gliadin peptide (P31-43) treated Caco-2 cells. Results show that cells treated with PP2, PP5, and PP6 peptides significantly reduce the cell mortality caused by P31-43. Three PPs significantly reduce the P31-43-induced decrease in ROS levels to control levels, and there is no difference between them and the vitamin C (Vc) group. The results in terms of antioxidant-related enzymes show that PPs significantly decrease superoxide dismutase activity (SOD), glutathione reductases (GR), and glutathione (GSH)/oxidized glutathione (GSSG) levels, thus significantly enhancing the antioxidant level of cells. By studying the key proteins of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) pathway, it is found that PPs activate the Keap1/Nrf2 signaling pathway. CONCLUSION: The study finds that peptides from peas can effectively alleviate ɑ-gliadin peptide-induced cell damage. The discovery of these food-derived peptides provides novel potential solutions for the prevention and treatment of CD.


Assuntos
Gliadina , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Transdução de Sinais , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Gliadina/farmacologia , Humanos , Células CACO-2 , Transdução de Sinais/efeitos dos fármacos , Antioxidantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Cacau/química , Peptídeos/farmacologia , Pisum sativum/química , Estresse Oxidativo/efeitos dos fármacos , Glutationa/metabolismo , Glutationa/farmacologia , Proteínas de Ervilha/farmacologia , Superóxido Dismutase/metabolismo , Doença Celíaca/prevenção & controle , Doença Celíaca/tratamento farmacológico
2.
Amino Acids ; 55(11): 1601-1619, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37803248

RESUMO

Enzyme therapy for celiac disease (CeD), which digests gliadin into non-immunogenic and non-toxic peptides, can be an appropriate treatment option for CeD. Here, we have investigated the effectiveness of bromelain and ficin on gliadin digestion using in vitro, such as SDS-PAGE, HPLC, and circular dichroism (CD). Furthermore, the cytotoxicity of gliadin and 19-mer peptide before and after digestion with these enzymes was evaluated using the MTT assay in the Caco-2 cell line. Finally, we examined the effect of these treatments along with Larazotide Acetate on the expression of genes involved in cell-tight junctions, such as Occludin, Claudin 3, tight junction protein-1, and Zonulin in the Caco-2 cell line. Our study demonstrated bromelain and ficin digestion effects on the commercial and wheat-extracted gliadin by SDS-PAGE, HPLC, and CD. Also, the cytotoxicity results on Caco-2 showed that toxicity of the gliadin and synthetic 19-mer peptide was decreased by adding bromelain and ficin. Furthermore, the proteolytic effects of bromelain and ficin on gliadin indicated the expression of genes involved in cell-tight junctions was improved. This study confirms that bromelain and ficin mixture could be effective in improving the symptoms of CeD.


Assuntos
Doença Celíaca , Gliadina , Humanos , Células CACO-2 , Gliadina/farmacologia , Gliadina/metabolismo , Junções Íntimas , Ficina , Bromelaínas/farmacologia , Peptídeos/farmacologia
3.
Methods Cell Biol ; 179: 13-20, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37625871

RESUMO

Celiac disease is an autoimmune response to gluten proteins. While causes for celiac disease have been identified, there is no effective treatment other than diet control. In vitro models for celiac disease are important for quickly gaining understanding of the disease mechanism and testing potential treatments. Here we describe an ex vivo stimulation of intestinal epithelial cells with gliadin peptides as a method to induce celiac disease features in vitro.


Assuntos
Doença Celíaca , Gliadina , Humanos , Gliadina/farmacologia , Intestinos , Células Epiteliais
4.
Cell Rep ; 39(11): 110956, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35705047

RESUMO

Celiac disease (CD) is a multisystem disease in which different organs may be affected. We investigate whether circulating innate lymphoid cells (ILCs) contribute to the CD peripheral inflammatory status. We find that the CD cytokine profile is characterized by high concentrations of IL-12p40, IL-18, and IFN-γ, paralleled by an expansion of ILC precursors (ILCPs). In the presence of the gliadin peptides p31-43 and pα-9, ILCPs from CD patients increase transglutaminase 2 (TG2) expression, produce IL-18 and IFN-γ, and stimulate CD4+ T lymphocytes. IFN-γ is also produced upon stimulation with IL-12p40 and IL-18 and is inhibited by the addition of vitamin D. Low levels of blood vitamin D correlate with high IFN-γ and ILCP presence and mark the CD population mostly affected by extraintestinal symptoms. Dietary vitamin D supplementation appears to be an interesting therapeutic approach to dampen ILCP-mediated IFN-γ production.


Assuntos
Doença Celíaca , Imunidade Inata , Doença Celíaca/imunologia , Doença Celíaca/metabolismo , Gliadina/metabolismo , Gliadina/farmacologia , Humanos , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Linfócitos/metabolismo , Vitamina D/metabolismo , Vitamina D/farmacologia
5.
J Steroid Biochem Mol Biol ; 220: 106083, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35257869

RESUMO

Previous studies have shown a relationship between vitamin D and celiac disease (CD), however little evidence is available examining the direct effects of vitamin D on pathological features of this disease. In this study we evaluated the effect of oral administration of different doses of native vitamin D3 (cholecalciferol) in enteropathic mice. Female non-obese diabetic (NOD)/ShiLt.J mice were fed standard or gluten-free diet and administered gliadin (5 µg/kg) to induce a celiac pathology. Healthy control (gluten-free diet, without gliadin) and control for pathology (standard diet, with gliadin) were administered olive oil. All other experimental groups received gliadin and standard diet plus oral cholecalciferol (5, 10, 20, 50 and 130 µg/kg). Serum levels of 25(OH)D3, calcium and zonulin and expression of vitamin D receptor (VDR), CD3 and zonula occludens-1 (ZO-1) by immunohistochemistry as well as intestinal histological and histomorphometric analyses were undertaken. Although no difference in serum levels of 25(OH)D3, calcium or zonulin was observed in cholecalciferol-treated mice vs. healthy controls, a significant improvement in intestinal mucosa pathological features in mice administered cholecalciferol was observed by histological analysis. Villi length was also significantly increased by cholecalciferol in a dose-dependent manner. Immunohistochemical staining revealed increased expression of CD3 and ZO-1 in celiac mice compared to mice receiving high dose (130 µg/kg) cholecalciferol. These findings show the effect of oral cholecalciferol on signature features of CD in a mouse model of CD. Further dose-ranging studies to investigate the efficacy of cholecalciferol for the treatment of CD are warranted.


Assuntos
Doença Celíaca , Colecalciferol , Animais , Calcifediol , Cálcio , Cálcio da Dieta , Doença Celíaca/tratamento farmacológico , Colecalciferol/farmacologia , Colecalciferol/uso terapêutico , Modelos Animais de Doenças , Feminino , Gliadina/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Vitamina D
6.
J Cancer Res Ther ; 17(6): 1445-1453, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34916376

RESUMO

BACKGROUND: Azoxymethane (AOM) is a potent carcinogenic agent commonly used to induce colon cancer in rats and mice, with the cytotoxicity of AOM mediated by oxidative stress. AIM OF STUDY: This study investigated the protective effect of a natural antioxidant (GliSODin) against AOM-induced oxidative stress and carcinogenesis in rat colon. METHODS: Twenty male Wistar rats were randomly divided into four groups (five rats/group). The control group was fed a basal diet. AOM-treated group (AOM) was fed a basal diet and received intraperitoneal injections of AOM for 2 weeks at a dose of 15 mg/kg. The GliSODin treatment group (superoxide dismutase [SOD]) received oral supplementation of GliSODin (300 mg/kg) for 3 months, and the fourth combined group received AOM and GliSODin (AOM + SOD). All animals were continuously fed ad libitum until the age of 16 weeks when all rats were sacrificed. The colon tissues were examined microscopically for pathological changes and aberrant crypt foci (ACF) development, oxidant status (lipid peroxidation-LPO), and enzyme antioxidant system (glutathione [GSH], GSH-S-transferase, catalase, and SOD). RESULTS: Our results showed that AOM induced ACF development and oxidative stress (GSH depletion and lipid peroxidation) in rat colonic cells. The concomitant treatment of AOM with GliSODin significantly ameliorated the cytotoxic effects of AOM. CONCLUSION: The results of this study provide in vivo evidence that GliSODin reduced the AOM-induced colon cancer in rats, through their potent antioxidant activities.


Assuntos
Antioxidantes/farmacologia , Neoplasias do Colo/tratamento farmacológico , Gliadina/farmacologia , Proteínas de Plantas/farmacologia , Superóxido Dismutase/farmacologia , Animais , Antioxidantes/uso terapêutico , Azoximetano/toxicidade , Carcinogênese/induzido quimicamente , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Colo/efeitos dos fármacos , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/patologia , Cucurbitaceae/enzimologia , Ensaios de Seleção de Medicamentos Antitumorais , Gliadina/uso terapêutico , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Plantas/uso terapêutico , Ratos , Superóxido Dismutase/uso terapêutico , Triticum/química
7.
Sci Rep ; 11(1): 10373, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990672

RESUMO

The development of inorganic antibacterial agents that impart antibacterial properties to biomaterials has attracted wide attention. The paper introduced a kind of hybrid nanosphere antibacterial agent composed of wheat gliadin (WG) and zinc oxide (ZnO), with antibacterial efficacy and low toxicity. The ZnO/WG hybrid nanospheres were environment-friendly integrated by molecular self-assembly co-precipitating and freeze-drying transformation, and were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), atomic absorption spectroscopy (AAS), specific surface and pore size analysis, bacteriostasis test, reactive oxygen species (ROS) determination and safety evaluation. It was found that the prepared hybrid nanospheres were composed of two components, WG and ZnO, with a diameter scope of 100-200 nm; the content of ZnO in the hybrid nanospheres can reach 46.9-70.2% (w/w); the bacteriostasis tests proved that the prepared ZnO/WG nanospheres generating ROS, have a significant inhibitory effect on E. coli and S. aureus; furthermore, the ZnO/WG nanospheres are relatively safe and highly biocompatible in cells and mice. Therefore, the prepared novel ZnO/WG hybrid nanospheres were supposed to apply in the preparation of anti-infective wound dressings, tissue engineering skin scaffold materials, food, and cosmetics preservatives, and so on.


Assuntos
Antibacterianos/farmacologia , Composição de Medicamentos/métodos , Gliadina/farmacologia , Óxido de Zinco/farmacologia , Administração Oral , Animais , Antibacterianos/química , Escherichia coli/efeitos dos fármacos , Feminino , Liofilização , Gliadina/química , Química Verde , Hemólise/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Nanosferas/química , Staphylococcus aureus/efeitos dos fármacos , Testes de Toxicidade Subaguda , Triticum/química , Óxido de Zinco/química
8.
Int J Mol Sci ; 22(1)2020 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-33375311

RESUMO

There is a need to assess the relationship between improved rheological properties and the immunogenic potential of wheat proteins. The present study aimed to investigate the in vitro effects of total protein extracts from three modern and two landrace Triticum aestivum commercial flour mixes, with significant differences in gluten strength (GS), on cell lines. Cytotoxicity and innate immune responses induced by wheat proteins were investigated using Caco-2 monocultures, two dimensional (2D) Caco-2/U937 co-cultures, and three dimensional (3D) co-cultures simulating the intestinal mucosa with Caco-2 epithelial cells situated above an extra-cellular matrix containing U937 monocytes and L929 fibroblasts. Modern wheat proteins, with increased GS, significantly reduced Caco-2 cell proliferation and vitality in monoculture and 2D co-cultures than landrace proteins. Modern wheat proteins also augmented Caco-2 monolayer disruption and tight junction protein, occludin, redistribution in 3D co-cultures. Release of interleukin-8 into the cell medium and increased U937 monocyte migration in both 2D and 3D co-cultures were similarly apparent. Immuno-activation of migrating U937 cells was evidenced from cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. The modern wheat proteins, with gluten polymorphism relatedness and increased GS, were shown to be more cytotoxic and immunogenic than the landrace wheat proteins.


Assuntos
Gliadina/farmacologia , Glutens/farmacologia , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Extratos Vegetais/farmacologia , Triticum/química , Humanos , Técnicas In Vitro , Mucosa Intestinal/efeitos dos fármacos
9.
Nutrients ; 12(3)2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32120967

RESUMO

Celiac disease (CD) presents as chronic low-grade inflammation of the small intestine often characterized by psychiatric comorbidities. The brain-derived neurotrophic factor (BDNF), which we have shown to be reduced in the serum of CD patients, acts as the bridge between immune activation and the nervous system adaptive response. Since Lactobacillus has been shown to upregulate BDNF, this study aimed to evaluate whether the administration of Lactobacillus rhamnosus GG (L.GG) could positively affect the brain BDNF system in rats mimicking the CD lesions. Data have shown that the administration of pepsin-trypsin digested gliadin (PTG) and L.GG alter the levels of mature BDNF (mBDNF), as evaluated by Western blotting. PTG provoked a reduction of mBDNF compared to controls, and a compensatory increase of its receptor TrkB. L.GG induced a slight positive effect on mBDNF levels under normal conditions, while it was able to rescue the PTG-induced reduced expression of mBDNF. The curative effect of L.GG was finely tuned, accompanied by the reduction of TrkB, probably to avoid the effect of excessive BDNF.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Encéfalo , Doença Celíaca , Gliadina/efeitos adversos , Lacticaseibacillus rhamnosus , Receptor trkB/biossíntese , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Doença Celíaca/induzido quimicamente , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Gliadina/química , Gliadina/farmacologia , Pepsina A , Ratos , Ratos Wistar , Tripsina
10.
Mol Nutr Food Res ; 64(6): e1900989, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31970917

RESUMO

SCOPE: Circulating dendritic cell (DC) and monocyte subsets contribute to the pool of intestinal DC and macrophages in celiac disease (CeD), an autoimmune gut disorder triggered by dietary gluten. Here, this study aims to characterize these circulating subsets in CeD and assess the effect of different gliadin-derived peptides on conventional DC (cDC). METHODS AND RESULTS: Flow cytometry profiling of peripheral blood mononuclear cells reveals a slight decrease in the proportion of plasmacytoid and type 1 cDC in gluten-free diet (GFD)-treated CeD patients. In comparison to healthy donors, DC and monocyte subsets from active and GFD-treated CeD patients display an increased gut-homing profile. Type 2 cDC (cDC2) are sorted and stimulated with the gliadin-derived peptides 8-mer, 19-mer, and 33-mer. All peptides induce cDC2 maturation, although the profile is different. While peptide 8-mer induces a Th1/Th17 pro-inflammatory cytokine profile in active CeD patients, cDC2 primed with peptide 33-mer displays a higher capacity to promote gut-homing CCR9+ expression onto autologous T-cells. CONCLUSION: Distinct gliadin-derived peptides elicit different effects on cDC2 phenotype and function. This effect is compatible with a model where diverse gliadin peptides may cooperate to promote full cDC2 activation and the subsequent T-cell response in genetically predisposed individuals.


Assuntos
Doença Celíaca/patologia , Células Dendríticas/fisiologia , Gliadina/farmacologia , Adulto , Antígenos CD/metabolismo , Estudos de Casos e Controles , Doença Celíaca/dietoterapia , Doença Celíaca/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Dieta Livre de Glúten , Feminino , Trato Gastrointestinal/metabolismo , Gliadina/química , Gliadina/metabolismo , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/farmacologia , Receptores CCR/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
11.
FEBS J ; 287(10): 2134-2149, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31659864

RESUMO

Celiac disease (CeD) is a highly prevalent chronic immune-mediated enteropathy developed in genetically predisposed individuals after ingestion of a group of wheat proteins (called gliadins and glutenins). The 13mer α-gliadin peptide, p31-43, induces proinflammatory responses, observed by in vitro assays and animal models, that may contribute to innate immune mechanisms of CeD pathogenesis. Since a cellular receptor for p31-43 has not been identified, this raises the question of whether this peptide could mediate different biological effects. In this work, we aimed to characterize the p31-43 secondary structure by different biophysical and in silico techniques. By dynamic light scattering and using an oligomer/fibril-sensitive fluorescent probe, we showed the presence of oligomers of this peptide in solution. Furthermore, atomic force microscopy analysis showed p31-43 oligomers with different height distribution. Also, peptide concentration had a very strong influence on peptide self-organization process. Oligomers gradually increased their size at lower concentration. Whereas, at higher ones, oligomers increased their complexity, forming branched structures. By CD, we observed that p31-43 self-organized in a polyproline II conformation in equilibrium with ß-sheets-like structures, whose pH remained stable in the range of 3-8. In addition, these findings were supported by molecular dynamics simulation. The formation of p31-43 nanostructures with increased ß-sheet structure may help to explain the molecular etiopathogenesis in the induction of proinflammatory effects and subsequent damage at the intestinal mucosa in CeD.


Assuntos
Doença Celíaca/tratamento farmacológico , Gliadina/farmacologia , Imunidade Inata/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Células CACO-2 , Doença Celíaca/genética , Doença Celíaca/imunologia , Doença Celíaca/patologia , Gliadina/genética , Gliadina/imunologia , Gliadina/ultraestrutura , Humanos , Imunidade Inata/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Microscopia de Força Atômica , Conformação Molecular/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/ultraestrutura , Peptídeos/química , Peptídeos/imunologia , Peptídeos/farmacologia , Conformação Proteica em Folha beta , Soluções/química , Água/química
12.
Sci Rep ; 9(1): 7029, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31065051

RESUMO

Celiac disease (CD) is an immune-mediated disorder triggered by gluten exposure. The contribution of the adaptive immune response to CD pathogenesis has been extensively studied, but the absence of valid experimental models has hampered our understanding of the early steps leading to loss of gluten tolerance. Using intestinal organoids developed from duodenal biopsies from both non-celiac (NC) and celiac (CD) patients, we explored the contribution of gut epithelium to CD pathogenesis and the role of microbiota-derived molecules in modulating the epithelium's response to gluten. When compared to NC, RNA sequencing of CD organoids revealed significantly altered expression of genes associated with gut barrier, innate immune response, and stem cell functions. Monolayers derived from CD organoids exposed to gliadin showed increased intestinal permeability and enhanced secretion of pro-inflammatory cytokines compared to NC controls. Microbiota-derived bioproducts butyrate, lactate, and polysaccharide A improved barrier function and reduced gliadin-induced cytokine secretion. We concluded that: (1) patient-derived organoids faithfully express established and newly identified molecular signatures characteristic of CD. (2) microbiota-derived bioproducts can be used to modulate the epithelial response to gluten. Finally, we validated the use of patient-derived organoids monolayers as a novel tool for the study of CD.


Assuntos
Doença Celíaca/microbiologia , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal/citologia , Organoides , Adulto , Idoso , Doença Celíaca/genética , Doença Celíaca/patologia , Proliferação de Células , Citocinas/metabolismo , Duodeno/citologia , Duodeno/patologia , Disbiose/metabolismo , Microbioma Gastrointestinal/genética , Expressão Gênica , Gliadina/metabolismo , Gliadina/farmacologia , Glutens/metabolismo , Glutens/farmacologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Células-Tronco/patologia
13.
Cell Microbiol ; 21(8): e13035, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31042331

RESUMO

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N. flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N. flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD-N. flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L. paracasei-CBA reduced CD-N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N. flavescens induces metabolic imbalance in CaCo-2 cells, and the L. paracasei-CBA probiotic could be used to correct CD-associated dysbiosis.


Assuntos
Lacticaseibacillus paracasei/química , Mitocôndrias/efeitos dos fármacos , Neisseria/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Probióticos/farmacologia , Trifosfato de Adenosina/agonistas , Trifosfato de Adenosina/metabolismo , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/microbiologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Células CACO-2 , Doença Celíaca/metabolismo , Doença Celíaca/microbiologia , Doença Celíaca/terapia , Meios de Cultivo Condicionados/farmacologia , Disbiose/metabolismo , Disbiose/microbiologia , Disbiose/terapia , Expressão Gênica , Gliadina/antagonistas & inibidores , Gliadina/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Lacticaseibacillus paracasei/fisiologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Neisseria/genética , Neisseria/crescimento & desenvolvimento , Neisseria/patogenicidade , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
14.
J Pept Sci ; 25(5): e3161, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30912242

RESUMO

Inflammation of intestinal tissue in patients affected by celiac disease (CD) originates from the adaptive and innate immune responses elicited by the undigested gliadin fragments through molecular mechanisms not yet completely described. Undigested A-gliadin peptide P31-43 is central to CD pathogenesis, entering enterocytes in vesicular compartments by endocytosis and inducing an innate immune response in CD intestinal mucosa. This study focused on the reasons why P31-43 does not behave as adaptive immunogenic agent. Once obtained by NMR analysis, the three-dimensional model of P31-43 was used to implement a series of in silico experiments aimed to explore the ability of the peptide to interact with HLA-DQ2 and the corresponding receptor onto T cells. Our results show that P31-43 is a poor ligand for DQ2 and/or T-cell receptor. This study was also aimed to investigate, from a structural point of view, the previous experimental findings by which P31-43 is able to enhance the phosphorylation level of the protein ERK2, while some P31-43 Ala-mutants decrease or totally inhibit that process. The molecular models of P31-43, P31-43 P36A, and F37A mutants were used for in silico docking experiments onto the ERK2 structure. The experiments support the hypothesis that P31-43 F37A works as an ERK2 phosphorylation inhibitor because it binds to the ERK2 phosphorylation site. This study reports on the structural properties of so far never NMR characterized gliadin peptides relevant in CD and explores details about their mechanisms of action.


Assuntos
Doença Celíaca/imunologia , Gliadina/farmacologia , Imunidade Inata/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Gliadina/química , Humanos , Imunidade Inata/imunologia , Mucosa Intestinal/imunologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fosforilação
15.
Funct Integr Genomics ; 19(1): 123-136, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30159724

RESUMO

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.


Assuntos
Proteínas Arqueais/genética , Proteínas de Bactérias/genética , Glutens/metabolismo , Peptídeo Hidrolases/genética , Proteínas de Plantas/genética , Triticum/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Biolística , Doença Celíaca/dietoterapia , Doença Celíaca/imunologia , Chryseobacterium/enzimologia , Chryseobacterium/genética , Expressão Gênica , Engenharia Genética/métodos , Gliadina/imunologia , Gliadina/isolamento & purificação , Gliadina/metabolismo , Gliadina/farmacologia , Glutens/química , Glutens/imunologia , Hordeum/enzimologia , Hordeum/genética , Humanos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteólise , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , Transgenes , Triticum/enzimologia
16.
Dig Liver Dis ; 51(1): 47-54, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30055963

RESUMO

BACKGROUND: Gliadins are involved in gluten-related disorders and are responsible for the alteration of the cellular redox balance. It is not clear if the gliadin-related oxidative stress can induce DNA damage in enterocytes. AIM: To investigate any possible genotoxicity caused by gliadin and to assess its relationship with oxidative stress in vitro and ex vivo. METHODS: Caco-2 cells were exposed for 6-12-24 h to increasing concentrations (250 µg/mL-1000 µg/mL) of digested gliadin. We investigated: cytotoxicity, oxidative balance (reactive oxygen species, ROS), DNA damage (comet assay and γ-H2AX detection), transglutaminase type 2 (TG2) activity and annexin V expression. H2AX and 8-OHG immunohistochemistry has been evaluated on duodenal biopsies of celiac subjects and controls. RESULTS: Gliadin induced a significant increase (+50%) of ROS after 12 h of exposition starting with a 500 µg/mL dose of gliadin. Comet assay and γ-H2AX demonstrated DNA damage, evident at the gliadin concentration of 500 µg/mL after 24 h. TG2 activity increased in chromatin and cytoskeleton cellular compartments at different gliadin doses (250/500/1000 µg/mL). The γ-H2AX and 8-OHG immunohistochemistry was altered in the duodenal biopsies of celiac patients. CONCLUSIONS: Gliadin induces cellular oxidative stress, DNA damage and pro-apoptotic stimulation in Caco-2 cells and in the duodenal mucosa of celiac patients.


Assuntos
Doença Celíaca/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Gliadina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Apoptose , Western Blotting , Células CACO-2/efeitos dos fármacos , Ensaio Cometa , Enterócitos/efeitos dos fármacos , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade
17.
EMBO J ; 38(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30498130

RESUMO

Intestinal handling of dietary proteins usually prevents local inflammatory and immune responses and promotes oral tolerance. However, in ~ 1% of the world population, gluten proteins from wheat and related cereals trigger an HLA DQ2/8-restricted TH1 immune and antibody response leading to celiac disease. Prior epithelial stress and innate immune activation are essential for breaking oral tolerance to the gluten component gliadin. How gliadin subverts host intestinal mucosal defenses remains elusive. Here, we show that the α-gliadin-derived LGQQQPFPPQQPY peptide (P31-43) inhibits the function of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel pivotal for epithelial adaptation to cell-autonomous or environmental stress. P31-43 binds to, and reduces ATPase activity of, the nucleotide-binding domain-1 (NBD1) of CFTR, thus impairing CFTR function. This generates epithelial stress, tissue transglutaminase and inflammasome activation, NF-κB nuclear translocation and IL-15 production, that all can be prevented by potentiators of CFTR channel gating. The CFTR potentiator VX-770 attenuates gliadin-induced inflammation and promotes a tolerogenic response in gluten-sensitive mice and cells from celiac patients. Our results unveil a primordial role for CFTR as a central hub orchestrating gliadin activities and identify a novel therapeutic option for celiac disease.


Assuntos
Doença Celíaca/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Gliadina/farmacologia , Fragmentos de Peptídeos/farmacologia , Adolescente , Aminofenóis/administração & dosagem , Aminofenóis/farmacologia , Animais , Células CACO-2 , Doença Celíaca/tratamento farmacológico , Doença Celíaca/genética , Linhagem Celular , Criança , Regulador de Condutância Transmembrana em Fibrose Cística/química , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Masculino , Camundongos , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Domínios Proteicos , Quinolonas/administração & dosagem , Quinolonas/farmacologia , Adulto Jovem
18.
Biochem Biophys Res Commun ; 503(3): 2139-2145, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30097270

RESUMO

Clinical attention to gluten-related disorders, such as celiac disease and nonceliac gluten sensitivity, is on the rise. However, identifying the pathophysiological mechanisms of gluten-related disorders remains elusive. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. Caenorhabditis elegans has been widely used as the predominant experimental animal model to study toxicity and stress response in biomedical research. We investigated the stress response induced by gliadin intake in C. elegans to evaluate its toxicity and found brood size, body bending, and pumping rates to be significantly altered in response to gliadin. Notably, reactive oxygen species (ROS) production and Pgst-4::GFP transgene expression, an indicator of the oxidative-stress response, were significantly increased after gliadin intake. Reduced pumping rates were most likely caused by gliadin-induced oxidative stress, since pumping rates in oxidative stress-sensitive mev-1 mutants were more severely reduced than in oxidative stress-resistant daf-2 mutants following gliadin intake. Our results indicated that gluten/gliadin intake in C. elegans triggered ROS production and induced an oxidative stress response that reduced pumping rates and decreased brood size. We suggest C. elegans to be a useful model system for studying gluten/gliadin toxicity.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Gliadina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ração Animal , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Relação Dose-Resposta a Droga , Gliadina/metabolismo , Locomoção/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
19.
Sci Rep ; 8(1): 10821, 2018 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018339

RESUMO

Celiac disease (CD) is an autoimmune disease characterized by inflammation of the intestinal mucosa due to an immune response to wheat gliadins. Some gliadin peptides are resistant to intestinal digestion (e.g., A-gliadin P31-43) and induce a stress/innate immune response, but the reason why they are dangerous in the intestines of patients with CD is unknown. In the present study, P31-43 activated IFN-α, a mediator of the innate immune response in CD, in the intestine of subjects with CD and an enterocyte cell line, CaCo-2. P31-43 cooperated with a viral ligand to activate the TLR7 pathway by interfering with endocytic trafficking. Based on these results, the vesicular pathway regulates the innate/inflammatory response to viral ligands and bioactive dietary peptides. Suggesting that together with viral infections, alimentary proteins able to mimic and potentiate the innate immune response to viruses, can trigger an autoimmune disease such as CD.


Assuntos
Doença Celíaca/patologia , Endocitose/efeitos dos fármacos , Gliadina/farmacologia , Imunidade Inata/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Adolescente , Células CACO-2 , Doença Celíaca/imunologia , Criança , Pré-Escolar , Dieta Livre de Glúten , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Feminino , Gliadina/química , Guanosina/análogos & derivados , Guanosina/farmacologia , Humanos , Interferon-alfa/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Proteínas de Resistência a Myxovirus/metabolismo , NF-kappa B/metabolismo , Fragmentos de Peptídeos/química , Transdução de Sinais/efeitos dos fármacos , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo
20.
Int J Mol Sci ; 19(2)2018 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-29473905

RESUMO

Gliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for celiac disease (CD), and its toxicity is not reduced by digestion with gastro-pancreatic enzymes. Importantly, it is proved that an innate immunity to gliadin plays a key role in the development of CD. The immune response induces epithelial stress and reprograms intraepithelial lymphocytes into natural killer (NK)-like cells, leading to enterocyte apoptosis and an increase in epithelium permeability. In this contribution, we have reported that in Caco-2 cells the administration of enzymatically digested gliadin (PT-gliadin) reduced significantly the expression of the autophagy-related marker LC3-II. Furthermore, electron and fluorescent microscope analysis suggested a compromised functionality of the autophagosome apparatus. The rescue of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was obtained with a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce a significant improvement in the clearance of extra- and intra-cellular fluorescent PT-gliadin amount. Altogether, our results highlighted the possible contribution of the autophagy process in the degradation and in the reduction of extra-cellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD.


Assuntos
Autofagia/efeitos dos fármacos , Gliadina/farmacologia , Pepsina A/metabolismo , Tripsina/metabolismo , Apoptose/efeitos dos fármacos , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Fluorescência , Gliadina/ultraestrutura , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Agregados Proteicos/efeitos dos fármacos
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