RESUMO
BACKGROUND: Total parenteral nutrition (TPN), a necessary treatment for patients who cannot receive enteral nutrition, is associated with infectious complications due in part to a loss of intestinal epithelial barrier function (EBF). Using a mouse model of TPN, with enteral nutrient deprivation, we previously demonstrated an increase in mucosal interferon-γ and tumor necrosis factor-α; these cytokine changes are a major mediator driving a reduction in epithelial tight junction (TJ) protein expression. However, the exact ultrastructural changes to the intestinal epithelial barrier have not been previously described. AIM: We hypothesized that TPN dependence results in ultrastructural changes in the intestinal epithelial TJ meshwork. METHODS: C57BL/6 mice underwent internal jugular venous cannulation and were given enteral nutrition or TPN with enteral nutrient deprivation for 7 days. Freeze-fracture electron microscopy was performed on ileal tissue to characterize changes in TJ ultrastructure. EBF was measured using transepithelial resistance and tracer permeability, while TJ expression was measured via Western immunoblotting and immunofluorescence staining. RESULTS: While strand density, linearity, and appearance were unchanged, TPN dependence led to a mean reduction in one horizontal strand out of the TJ compact meshwork to a more basal region, resulting in a reduction in meshwork depth. These findings were correlated with the loss of TJ localization of claudin-4 and tricellulin, reduced expression of claudin-5 and claudin-8, and reduced ex vivo EBF. CONCLUSION: Tight junction ultrastructural changes may contribute to reduced EBF in the setting of TPN dependence.
Assuntos
Mucosa Intestinal/citologia , Nutrição Parenteral Total/efeitos adversos , Junções Íntimas/ultraestrutura , Animais , Técnica de Fratura por Congelamento , Mucosa Intestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica/métodos , Junções Íntimas/efeitos dos fármacosRESUMO
Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Intestinos/citologia , Intestinos/virologia , Espaço Intracelular/virologia , Pinocitose , Replicação Viral , Transporte Biológico , Células CACO-2 , Citoesqueleto/virologia , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Humanos , Intestinos/ultraestrutura , Virulência , Internalização do VírusRESUMO
Protein fermentation end products may damage the colonic mucosa, which could be counteracted by dietary inclusion of fermentable carbohydrates (fCHO). Although fermentable crude protein (fCP) and fCHO are known to affect microbial ecology, their interactive effects on epithelial barrier function are unknown. In the present study, in a 2 × 2 factorial experiment, thirty-two weaned piglets were fed low-fCP/low-fCHO (14·5 % crude protein (CP)/14·5 % total dietary fibre (TDF)), low-fCP/high-fCHO (14·8 % CP/16·6 % TDF), high-fCP/low-fCHO (19·8 % CP/14·5 % TDF) and high-fCP/high-fCHO (20·1 % CP/18·0 % TDF) diets. After 21-23 d, samples of proximal and distal colonic mucosae were investigated in Ussing chambers with respect to the paracellular and transcytotic passages of macromolecules and epithelial ion transport. The high-fCHO diets were found to reduce the permeability of the distal colon to the transcytotic marker horseradish peroxidase (HRP, 44 kDa; P <0·05) and also reduce the paracellular permeation of N-hydroxysuccinimide-biotin into the submucosa (443 Da; P <0·05), whereas that of HRP was decreased by the high-fCP diets (P <0·01). Short-circuit current (active ion transport), transepithelial resistance (barrier function) and charge selectivity were largely unaffected in both the segments. However, the high-fCP diets were found to suppress the aldosterone-induced epithelial Na channel activity (P <0·01) irrespective of fCHO inclusion. The high-fCP diets generally reduced the expression of colonic claudin-1, claudin-2 and claudin-3 (P <0·01), while that of claudin-4 was increased by the high-fCHO diets (P <0·01). The high-fCHO diets also altered the ratio between occludin forms (P <0·05) and increased the expression of tricellulin in the proximal colon, which was not observed with high-fCP diets. In conclusion, dietary fCHO and fCP exerted few and largely independent effects on functional measurements, but altered tight junction protein composition in a compensatory way, so that colonic transport and barrier properties were only marginally affected.
Assuntos
Colo/fisiologia , Fibras na Dieta/administração & dosagem , Proteínas Alimentares/administração & dosagem , Fermentação , Sus scrofa/fisiologia , Proteínas de Junções Íntimas/análise , Animais , Colo/química , Dieta/veterinária , Feminino , Absorção Intestinal , Mucosa Intestinal/fisiologia , MasculinoRESUMO
AIMS: To further explore the anti-inflammatory properties of d-Limonene. MAIN METHODS: A rat model was used to compare evolution of TNBS (2,5,6-trinitrobenzene sulfonic acid)-induced colitis after oral feeding with d-Limonene compared to ibuprofen. Peripheral levels of TNF-α (Tumor Necrosis Factor alpha) were assessed in all animals. Cell cultures of fibroblasts and enterocytes were used to test the effect of d-Limonene respectively on TNFα-induced NF-κB (nuclear factor-kappa B) translocation and epithelial resistance. Finally, plasmatic inflammatory markers were examined in an observational study of diet supplementation with d-Limonene-containing orange peel extract (OPE) in humans. KEY FINDINGS: Administered per os at a dose of 10mg/kg p.o., d-Limonene induced a significant reduction of intestinal inflammatory scores, comparable to that induced by ibuprofen. Moreover, d-Limonene-fed rats had significantly lowered serum concentrations of TNF-α compared to untreated TNBS-colitis rats. The anti-inflammatory effect of d-Limonene also involved inhibition of TNFα-induced NF-κB translocation in fibroblast cultures. The application of d-Limonene on colonic HT-29/B6 cell monolayers increased epithelial resistance. Finally, inflammatory markers, especially peripheral IL-6, markedly decreased upon OPE supplementation of elderly healthy subjects submitted or not to 56 days of dietary supplementation with OPE. SIGNIFICANCE: In conclusion, d-Limonene indeed demonstrates significant anti-inflammatory effects both in vivo and in vitro. Protective effects on the epithelial barrier and decreased cytokines are involved, suggesting a beneficial role of d-Limonene as diet supplement in reducing inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Colite/tratamento farmacológico , Colite/patologia , Cicloexenos/administração & dosagem , Suplementos Nutricionais , Terpenos/administração & dosagem , Administração Oral , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Células HT29 , Humanos , Limoneno , Masculino , Camundongos , Ratos , Ratos WistarRESUMO
In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR), the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR). GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC) was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc)) in Ussing chambers. Endiandrin A (End A) has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF)-α. This was in part due to up-regulation of ß- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK) activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk)-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of ß- and γ-ENaC-subunits along with induction of sgk-1.
Assuntos
Colo/metabolismo , Ciclobutanos/farmacologia , Canais Epiteliais de Sódio/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Lauraceae/química , Lignanas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Glucocorticoides/agonistas , Animais , Ciclobutanos/química , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Glucocorticoides/farmacologia , Células HT29 , Humanos , Inflamação/metabolismo , Inflamação/patologia , Absorção Intestinal/efeitos dos fármacos , Lignanas/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Glucocorticoides/metabolismo , Sódio/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
TGFß (isoforms 1-3) has barrier-protective effects in the intestine. The mechanisms involved in regulating tight junction protein expression are poorly understood. The aim of this study was to elucidate TGFß-dependent protective effects with special attention to promoter regulation of tight junction proteins using the HT-29/B6 cell model. In addition, the effects of whey protein concentrate 1 (WPC1), a natural source of TGFß in human nutrition, were examined. For this purpose, the claudin-4 promoter was cloned and tested for its activity. It exhibited transactivation in response to TGFß1, which was intensified when Smad-4 was cotransfected, indicating a Smad-4-dependent regulatory component. Shortening and mutation of the promoter altered and attenuated this effect. WPC1 induced an increase in the claudin-4 protein level and resistance of HT-29/B6 cell monolayers. Anti-TGFß(1-3) antibodies blocked these whey protein effects, suggesting that a main part of this function was mediated through TGFß. This effect was observed on intact monolayers as well as when barrier function was impaired by preexposure to IFNγ. In conclusion, TGFß1 affects claudin-4 gene expression via Smad-4-dependent and -independent transcriptional regulation, resulting in barrier protection, a cytokine effect that is also found in whey protein concentrates used in enteral nutrition.
Assuntos
Mucosa Intestinal/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Leite/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Animais , Bovinos , Claudina-4 , Impedância Elétrica , Alimento Funcional , Genes Reporter , Células HT29 , Humanos , Interferon gama/toxicidade , Proteínas de Membrana/genética , Proteínas do Leite/uso terapêutico , Mutagênese Sítio-Dirigida , Mutação , Regiões Promotoras Genéticas , Substâncias Protetoras/metabolismo , Substâncias Protetoras/uso terapêutico , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes , Transdução de Sinais , Proteína Smad4/genética , Proteína Smad4/metabolismo , Ativação Transcricional , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/uso terapêutico , Proteínas do Soro do LeiteRESUMO
The epithelium in inflamed intestinal segments of patients with Crohn's disease is characterized by a reduction of tight junction strands, strand breaks, and alterations of tight junction protein content and composition. In ulcerative colitis, epithelial leaks appear early due to micro-erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin-2. Th1-cytokine effects by interferon-gamma in combination with TNFalpha are important for epithelial damage in Crohn's disease, while interleukin-13 (IL-13) is the key effector cytokine in ulcerative colitis stimulating apoptosis and upregulation of claudin-2 expression. Focal lesions caused by apoptotic epithelial cells contribute to barrier disturbance in IBD by their own conductivity and by confluence toward apoptotic foci or erosions. Another type of intestinal barrier defect can arise from alpha-hemolysin harboring E. coli strains among the physiological flora, which can gain pathologic relevance in combination with proinflammatory cytokines under inflammatory conditions. On the other hand, intestinal barrier impairment can also result from transcellular antigen translocation via an initial endocytotic uptake into early endosomes, and this is intensified by proinflammatory cytokines as interferon-gamma and may thus play a relevant role in the onset of IBD. Taken together, barrier defects contribute to diarrhea by a leak flux mechanism (e.g., in IBD) and can cause mucosal inflammation by luminal antigen uptake. Immune regulation of epithelial functions by cytokines may cause barrier dysfunction not only by tight junction impairments but also by apoptotic leaks, transcytotic mechanisms, and mucosal gross lesions.
Assuntos
Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Junções Íntimas/metabolismo , Animais , Apoptose , Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Epitélio/metabolismo , Humanos , Doenças Inflamatórias Intestinais/patologia , Interleucina-13/metabolismo , Mucosa Intestinal/patologiaRESUMO
OBJECTIVE: Backwash ileitis (BI) has not been identified as a risk factor for pouchitis. The aim of this study was to investigate the barrier function of the ileoanal pouch depending on the presence of BI. The incidence of pouchitis in a population of ulcerative colitis patients with BI is also reported. MATERIAL AND METHODS: Biopsies were taken from 80 patients with ulcerative colitis: a) terminal ileum prior to pouch creation (pre-IAP); b) 16 months after ileostomy closure (intact pouch); and c) during pouchitis. Patients were stratified into the BI group and the non-BI (ØBI) group. Barrier function was determined in Ussing-chambers as epithelial resistance by impedance analysis and as mannitol permeability from (3)H-mannitol fluxes. Na(+)-glucose co-transport was measured as a change in short-circuit current (I(SC)) after addition of glucose. Relative risk of developing pouchitis was calculated by corrected chi(2) test. RESULTS: In 13/21 (BI/ØBI) pre-IAP patients, 23/37 (BI/ØBI) with an intact pouch, and 35/7 (BI/ØBI) with pouchitis, epithelial resistance in BI/ØBI was 13.5+/-1.6/14.3+/-0.9 Omega.cm(2) for pre-IAP, 12.7+/-1.3/16.8+/-1.2 Omega x cm(2) (p<0.05 BI versus ØBI) for the intact pouch, and 10.1+/-1.1/9.9+/-1.8 Omega x cm(2) for pouchitis (p<0.05 BI versus ØBI with an intact pouch). No differences were found for electrogenic chloride secretion and active Na(+)-glucose co-transport between BI/ØBI in the three groups. In patients with BI, pouchitis was more common (35 versus 7 patients, odds ratio 33.0 (95% CI 8.3-143.9; p<0.0001)). CONCLUSIONS: Ulcerative colitis patients with BI show impaired barrier function in the further course of the ileoanal pouch. Thus, BI has a long-term impact on epithelial barrier function.
Assuntos
Colite Ulcerativa/cirurgia , Bolsas Cólicas/efeitos adversos , Ileíte/fisiopatologia , Mucosa Intestinal/metabolismo , Adulto , Bolsas Cólicas/fisiologia , Impedância Elétrica , Feminino , Humanos , Ileíte/etiologia , Mucosa Intestinal/fisiopatologia , Masculino , Manitol/farmacocinética , Permeabilidade , Pouchite/complicações , Pouchite/etiologia , Pouchite/fisiopatologia , Proctocolectomia Restauradora , Fatores de Risco , Proteínas de Transporte de Sódio-Glucose/metabolismoRESUMO
BACKGROUND AND AIMS: Bacterial overgrowth appears to play an important role in the pathogenesis of ileoanal pouches. Therefore, the capability of bacterial permeation and its determinants is of great interest. The aim of this study was to examine bacterial permeation in the ileoanal pouch and to correlate the results with the degree of inflammation, the epithelial resistance, the mucosal transport function, and the age of the ileoanal pouches. MATERIALS AND METHODS: Biopsies were taken from 54 patients before colectomy (n = 13; preileal pouch-anal anastomosis [IPAA]), and closure of ileostomy (n = 7; deviation), <1 year after closure of ileostomy (n = 8; intact pouch I), >1 year after closure of ileostomy (n = 16; intact pouch II), in the case of pouchitis (n = 11), and in 11 controls. Tissues were mounted in a miniaturized Ussing chamber. Escherichia coli was added to the mucosal side of the Ussing chamber, and the permeation was proven by serosal presence of E. coli. Epithelial and subepithelial resistance was determined by transmural impedance analysis. Active Na-glucose cotransport and active Cl secretion were measured. Specimens were analyzed by fluorescent in situ hybridization with oligonucleotide probes targeting the bacterial 16s ribosomal RNA. The bacteria in and on the tissue were enumerated. RESULTS: Bacterial permeation occurred in 2 of 13 pre-IPAA, 2 of 7 deviations, 0 of 8 intact pouch I, 9 of 16 intact pouch II, 5 of 11 pouchitis specimens, and 0 of 11 ileum controls. The frequency of bacterial permeation in the intact pouch II group is higher than in the intact pouch I group (P < 0.001). Epithelial resistance, mannitol fluxes, electrogenic chloride secretion, sodium-glucose cotransport of the bacterially permeated specimens versus nonpermeated of the intact pouch II group, and the pouchitis group and subepithelial resistance remained unchanged. Intramural bacteria could be detected by fluorescence in situ hybridization mainly in long-lasting pouches, but there was no correlation with bacterial permeation. CONCLUSIONS: The long-lasting ileoanal pouch is associated with increased bacterial permeability. This is not correlated with a disturbed function of the pouch mucosa but could be a precursor of pouchitis.