Caraterização in vitro dos efeitos tóxicos da patulina na integridade do epitélio intestinal e potenciais efeitos protetores da cisteína

A mucosa intestinal é a primeira barreira biológica encontrada pelas micotoxinas presentes nos alimentos, sendo a patulina, uma micotoxina produzida por fungos do género Penicillium spp., uma preocupação particular atendendo a que a exposição humana a esta micotoxina pode conduzir a efeitos imunológicos, neurológicos e gastrointestinais. Considerando estes efeitos para a saúde, o presente estudo tem como objetivos a avaliação do efeito tóxico da exposição intestinal a patulina, bem como a determinação do potencial efeito protetor da coadministração de patulina e cisteína na membrana intestinal, utilizando para o efeito células Caco-2. A integridade da membrana intestinal foi determinada pela medição da resistência elétrica transepitelial (TEER). Os resultados evidenciaram um decréscimo acentuado nos valores de TEER após 24 horas de exposição celular a 95 μM de patulina. Para as concentrações mais reduzidas verificou-se uma redução máxima inferior a 25% após 24 horas de exposição. A coadministração de patulina (95 μM) e cisteína (40 μM) revelou um decréscimo nos valores de TEER. O tratamento com cisteína em concentrações superiores ( 400 μM) revelou efeito protetor da membrana intestinal, tendo em conta os valores de TEER. Estes resultados contribuem para uma avaliação do risco mais precisa associada à exposição a contaminantes alimentares.

Characterization of in vitro effects of patulin on intestinal epithelial and immune cells

The intestinal mucosa is the first biological barrier encountered by natural toxins, and could possibly be exposed to high amounts of dietary mycotoxins. Patulin (PAT), a mycotoxin produced by Penicillium spp. during fruit spoilage, is one of the best known enteropathogenic mycotoxins able to alter functions of the intestine (Maresca et al., 2008). This study evaluated the effects of PAT on barrier function of the gut mucosa utilizing the intestinal epithelial cell model Caco-2, and scrutinized immunomodulatory effects using human peripheral blood mononuclear cells (PBMC) and human blood monocyte-derived dendritic cells (moDCs) as test systems. PAT exposure reduced Caco-2 cell viability at concentrations above 12 mM. As expected, the integrity of a polarized Caco-2 monolayer was affected by PAT exposure, as demonstrated by a decrease in TER values, becoming more pronounced at 50 mM. No effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3 at 50 mM. However, the expression of zonula occludens-1 (ZO-1) and myosin light chain 2 (MLC2) declined. Also, levels of phospho-MLC2 (p-MLC2) increased after 24 h of exposure to 50 mM of PAT. T cell proliferation was highly sensitive to PAT with major effects for concentrations above 10 nM of PAT. The same conditions did not affect the maturation of moDC. PAT causes a reduction in Caco-2 barrier function mainly by perturbation of ZO-1 levels and the phosphorylation of MLC. Low doses of PAT strongly inhibited T cell proliferation induced by a polyclonal activator, but had no effect on the maturation of moDC. These results provide new information that strengthens the concept that the epithelium and immune cells of the intestinal mucosa are important targets for the toxic effects of food contaminants like mycotoxins

The Role of Mucosal Defense in Intestinal Injury of Infants With Fetal Growth Retardation

Background: Infants with fetal growth retardation (FGR) are prone to intestinal disorders. Objectives: Aim of the study was to determine the role of mucosal defense ability in formation of gut injury in infants with FGR. Materials and Methods: 44 premature infants who were admitted to the Neonatal Intensive Care Unit were divided into two groups: 20 infants with FGR (FGR group) and 24 appropriate-for-gestational age newborns (AGA group). Control group consisted of 22 premature infants who were delivered after uncomplicated pregnancy. Gut barrier function was evaluated by detecting serum intestinal trefoil factor (ITF) and intestinal fatty acid binding protein (IFABP). The level of serum IFABP and ITF was measured by using ELISA method. Results: FGR group showed significantly higher ITF concentration than AGA group on the first days of life (P ˂ 0.01). High level of ITF in the FGR group significantly declines up to 7th - 10th day of life (P ˂ 0.01). This reduction was accompanied by increase of IFABP which is a marker of ischemic intestinal mucosal injury. Correlation analyses showed that ITF had a negative correlation with IFABP. Conclusions: Infants with fetal growth retardation are characterized by a high level of ITF on the first days of life. This protects intestinal mucosa under hypoxic conditions. Its subsequent decline accompanied by an increase of IFABP reflects the depletion of Goblet cells to secret ITF causing damage to the integrity of intestinal mucosal barrier.

Defective Apoptosis In Intestinal And Mesenteric Adipose Tissue Of Crohn's Disease Patients.

Crohn's disease (CD) is associated with complex pathogenic pathways involving defects in apoptosis mechanisms. Recently, mesenteric adipose tissue (MAT) has been associated with CD ethiopathology, since adipose thickening is detected close to the affected intestinal area. However, the potential role of altered apoptosis in MAT of CD has not been addressed. To evaluate apoptosis in the intestinal mucosa and MAT of patients with CD. Samples of intestinal mucosa and MAT from patients with ileocecal CD and from non-inflammatory bowel diseases patients (controls) were studied. Apoptosis was assessed by TUNEL assay and correlated with the adipocytes histological morphometric analysis. The transcriptional and protein analysis of selected genes and proteins related to apoptosis were determined. TUNEL assay showed fewer apoptotic cells in CD, when compared to the control groups, both in the intestinal mucosa and in MAT. In addition, the number of apoptotic cells (TUNEL) correlated significantly with the area and perimeter of the adipose cells in MAT. Transcriptomic and proteomic analysis reveal a significantly lower transcript and protein levels of Bax in the intestinal mucosa of CD, compared to the controls; low protein levels of Bax were found localized in the lamina propria and not in the epithelium of this tissue. Furthermore, higher level of Bcl-2 and low level of Caspase 3 were seen in the MAT of CD patients. The defective apoptosis in MAT may explain the singular morphological characteristics of this tissue in CD, which may be implicated in the pathophysiology of the disease.

Glutamine supplementation does not improve protein synthesis rate by the jejunal mucosa of the malnourished rat

It has been demonstrated that glutamine, a conditionally essential amino acid, improves nitrogen balance, acts as a stimulant of protein synthesis, and decreases proteolysis in myopathic children. In contrast, other studies have shown no beneficial effect of glutamine supplementation on burn victims or critically ill patients. Nonetheless, we hypothesized that glutamine supplementation would increase the fractional protein synthesis rate (FSR) in the jejunal mucosa of malnourished male Wistar rats. Thus, the objective of the present study was to test the effect of daily oral glutamine supplementation (0.42 g kg(-1) d(-1) for 14 days) on the FSR of the jejunal mucosa of healthy and malnourished rats. A 4-hour kinetic study with L-[1-(13)C]leucine was subsequently performed, and jejunal biopsies were obtained 1.5 cm from the Treitz angle and analyzed. Malnourished rats showed a 25% weight loss and increased urinary nitrogen excretion. Plasma amino acid concentration did not differ between groups. (13)C enrichment in plasma and jejunal cells was higher in the malnourished groups than in the healthy group. The FSR (percent per hour) was similar for the control and experimental groups (P > .05), with a mean range of 220%/h to 27%/h. Oral glutamine supplementation alone did not induce higher protein incorporation by the jejunal mucosa in malnourished rats, regardless of total food intake or the presence or absence of glutamine supplementation. (C) 2009 Elsevier Inc. All rights reserved.

Role of cytokines (TNF-alpha, IL-1 beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide

Introduction Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. Purpose Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11. Materials and methods Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-alpha and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and KC ELISA. Results CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-alpha, IL-1 beta and KC level and TNF-alpha immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-alpha, IL-1 beta and KC and TNF-alpha immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-alpha tissue level and TNF-alpha immuno-staining, but did not reduce the severity of diarrhea. Conclusion These results suggest an important role of TNF-alpha, IL-1 beta and KC in the pathogenesis of intestinal mucositis induced by CPT-11.

Heat stress impairs performance parameters, induces intestinal injury, and decreases macrophage activity in broiler chickens

Studies on environmental consequences of stress on animal production have grown substantially in the last few years for economic and animal welfare reasons. Physiological, hormonal, and immunological deficits as well as increases in animals` susceptibility to diseases have been reported after different stressors in broiler chickens. The aim of the current experiment is to describe the effects of 2 different heat stressors (31 +/- 1 and 36 +/- 1 degrees C/10 h per d) applied to broiler chickens from d 35 to 42 of life on the corticosterone serum levels, performance parameters, intestinal histology, and peritoneal macrophage activity, correlating and discussing the obtained data under a neuroimmune perspective. In our study, we demonstrated that heat stress (31 +/- 1 and 36 +/- 1 degrees C) increased the corticosterone serum levels and decreased BW gain and food intake. Only chickens submitted to 36 +/- 1 degrees C, however, presented a decrease in feed conversion and increased mortality. We also showed a decrease of bursa of Fabricius (31 +/- 1 and 36 +/- 1 degrees C), thymus (36 +/- 1 degrees C), and spleen (36 +/- 1 degrees C) relative weights and of macrophage basal (31 +/- 1 and 36 +/- 1 degrees C) and Staphylococcus aureus-induced oxidative burst (31 +/- 1 degrees C). Finally, mild multifocal acute enteritis characterized by an increased presence of lymphocytes and plasmocytes within the jejunum`s lamina propria was also observed. The stress-induced hypothalamic-pituitary-adrenal axis activation was taken as responsible for the negative effects observed on the chickens` performance and immune function and also the changes of the intestinal mucosa. The present obtained data corroborate with others in the field of neuroimmunomodulation and open new avenues for the improvement of broiler chicken welfare and production performance.

Dietary supplementation with vitamin E modulates avian intestinal immunity

The effect of dietary vitamin E on immunoglobulin A (IgA) antibody production, which acts as the first line of defence at the intestinal mucosa, has not been evaluated in chickens. In the present study the impact of the inclusion of supplementary levels of vitamin E to the diet, on total and antigen-specific IgA antibody titres, T-cell subsets and Ia+ cells, was assessed. From hatching, chickens received a maize-based diet which was supplemented with either 25, 250, 2500 or 5000 mg dl-alpha-tocopherol acetate/kg. Primary immunisation with tetanus toxoid (T. toxoid) emulsified in a vegetable oil-in-water adjuvant was administered by the intraperitoneal route at 21 d of age. At 35 d of age all birds received an oral booster vaccination of T. toxoid. Significantly higher total IgA antibody titres were present in the day 42 intestinal scrapings of birds receiving the 5000 mg/kg vitamin E-supplemented diet (VESD) (P=0.05) and a notable increase was observed in birds receiving the 250 mg/kg VESD (P=0.06). At days 21 and 42 total serum IgA antibody titres of birds receiving the 250 mg/kg VESD was significantly higher (P

Structural changes in the jejunal mucosa of mice infected with Schistosoma mansoni, fed low or high protein diets

The effects of high and low-protein diets on the structure of the jejunal mucosa were studied in Schistosoma mansoni infected mice (morphology and histomorphometry). Weaning male albino mice were infected with 80 cercariae, fed with high (20%) or low-protein (5%) diets and compared to uninfected controls under the same conditions. Mice were sacrificed 12 weeks after infection. Animals submitted to a low-protein diet showed lower weight curves, mainly when infected. In the jejunal mucosa, finger-like villi were the predominant pattern among uninfected high-protein fed animals, while the infected ones showed leaf-shaped and flattened villi in most cases. Undernourished infected mice had 65.7% leaf-shaped villi. A significant increase in the number of goblet cells was seen in infected mice. A decrease in the number of absorptive cells was detected in undernourished mice, particularly in infected ones.

Targeting of secretory IgA to Peyer's patch dendritic and T cells after transport by intestinal M cells.

In addition to being instrumental to the protection of mucosal epithelia, secretory IgA (SIgA) adheres to and is transported by intestinal Peyer's patch (PP) M cells. The possible functional reason for this transport is unknown. We have thus examined in mice the outcome of SIgA delivered from the intestinal lumen to the cells present in the underlying organized mucosa-associated lymphoreticular tissue. We show selective association of SIgA with dendritic cells and CD4(+) T and B lymphocytes recovered from PP in vitro. In vivo, exogenously delivered SIgA is able to enter into multiple PP lining the intestine. In PP, SIgA associates with and is internalized by dendritic cells in the subepithelial dome region, whereas the interaction with CD4(+) T cells is limited to surface binding. Interaction between cells and SIgA is mediated by the IgA moiety and occurs for polymeric and monomeric molecular forms. Thus, although immune exclusion represents the main function of SIgA, transport of the Ab by M cells might promote Ag sampling under neutralizing conditions essential to the homeostasis of mucosal surfaces.

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

Normal kinetics of intestinal glucose absorption in the absence of GLUT2: evidence for a transport pathway requiring glucose phosphorylation and transfer into the endoplasmic reticulum.

Glucose is absorbed through the intestine by a transepithelial transport system initiated at the apical membrane by the cotransporter SGLT-1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Here, we evaluated the impact of GLUT2 gene inactivation on this transepithelial transport process. We report that the kinetics of transepithelial glucose transport, as assessed in oral glucose tolerance tests, was identical in the presence or absence of GLUT2; that the transport was transcellular because it could be inhibited by the SGLT-1 inhibitor phlorizin, and that it could not be explained by overexpression of another known glucose transporter. By using an isolated intestine perfusion system, we demonstrated that the rate of transepithelial transport was similar in control and GLUT2(-/-) intestine and that it was increased to the same extent by cAMP in both situations. However, in the absence, but not in the presence, of GLUT2, the transport was inhibited dose-dependently by the glucose-6-phosphate translocase inhibitor S4048. Furthermore, whereas transport of [(14)C]glucose proceeded with the same kinetics in control and GLUT2(-/-) intestine, [(14)C]3-O-methylglucose was transported in intestine of control but not of mutant mice. Together our data demonstrate the existence of a transepithelial glucose transport system in GLUT2(-/-) intestine that requires glucose phosphorylation and transfer of glucose-6-phosphate into the endoplasmic reticulum. Glucose may then be released out of the cells by a membrane traffic-based pathway similar to the one we previously described in GLUT2-null hepatocytes.

Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) that overlies Peyer's patches (PPs) exhibits distinct features compared with the adjacent villus epithelium. Besides the presence of antigen-sampling membranous M cells and the down-regulation of digestive functions, it constitutively expresses the chemokine CCL20. The mechanisms that induce FAE differentiation and CCL20 expression are poorly understood. The aim of this work was to test whether lymphotoxin beta receptor signaling (LTbetaR), which plays a central role in PPs' organogenesis, mediates CCL20 gene expression in intestinal epithelial cells. METHODS: CCL20, lymphotoxin beta (LTbeta) and LTbetaR expression were monitored during embryonic development by in situ hybridization of mouse intestine. The human intestinal epithelial cell line T84 was used to study CCL20 expression following LTalpha(1)/beta(2) stimulation. In vivo CCL20 expression following agonistic anti-LTbetaR antibody treatment was studied by laser microdissection and quantitative RT-PCR. RESULTS: CCL20 was expressed in the FAE before birth at the time when the first hematopoietic CD4(+)CD3(-) appeared in the PP anlage. LTbetaR was expressed in the epithelium during PP organogenesis, making it a putative target for LTalpha(1)beta(2)signals. In vitro, CCL20 was induced in T84 cells upon LTbetaR signaling, either using an agonistic ligand or anti-LTbeta receptor agonistic antibody. LTalpha(1)beta(2)-induced CCL20 expression was found to be NF-kappaB dependent. LTbetaR signaling up-regulated CCL20 expression in the small intestinal epithelium in vivo. CONCLUSIONS: Our results show that LTbetaR signaling induces CCL20 expression in intestinal epithelial cells, suggesting that this pathway triggers constitutive production of CCL20 in the FAE.

Intestinal bacteria condition dendritic cells to promote IgA production.

Immunoglobulin (Ig) A represents the predominant antibody isotype produced at the intestinal mucosa, where it plays an important role in limiting the penetration of commensal intestinal bacteria and opportunistic pathogens. We show in mice that Peyer's Patch-derived dendritic cells (PP-DC) exhibit a specialized phenotype allowing the promotion of IgA production by B2 cells. This phenotype included increased expression of the retinaldehyde dehydrogenase 1 (RALDH1), inducible nitric oxide synthase (iNOS), B cell activating factor of the tumor necrosis family (BAFF), a proliferation-inducing ligand (APRIL), and receptors for the neuropeptide vasoactive intestinal peptide (VIP). The ability of PP-DC to promote anti-CD40 dependent IgA was partially dependent on retinoic acid (RA) and transforming growth factor (TGF)-beta, whilst BAFF and APRIL signaling were not required. Signals delivered by BAFF and APRIL were crucial for CD40 independent IgA production, although the contribution of B2 cells to this pathway was minimal. The unique ability of PP-DC to instruct naïve B cells to differentiate into IgA producing plasma cells was mainly imparted by the presence of intestinal commensal bacteria, and could be mimicked by the addition of LPS to the culture. These data indicate that exposure to pathogen-associated molecular patterns present on intestinal commensal bacteria condition DC to express a unique molecular footprint that in turn allows them to promote IgA production.

Conditioned polarized Caco-2 cell monolayers allow to discriminate for the ability of gut-derived microorganisms to modulate permeability and antigen-induced basophil degranulation.

BACKGROUND: Food allergy is a common allergic disorder--especially in early childhood. The avoidance of the allergenic food is the only available method to prevent further reactions in sensitized patients. A better understanding of the immunologic mechanisms involved in this reaction would help to develop therapeutic approaches applicable to the prevention of food allergy. OBJECTIVE: To establish a multi-cell in vitro model of sensitized intestinal epithelium that mimics the intestinal epithelial barrier to study the capacity of probiotic microorganisms to modulate permeability, translocation and immunoreactivity of ovalbumin (OVA) used as a model antigen. METHODS: Polarized Caco-2 cell monolayers were conditioned by basolateral basophils and used to examine apical to basolateral transport of OVA by ELISA. Activation of basophils with translocated OVA was measured by beta-hexosaminidase release assay. This experimental setting was used to assess how microorganisms added apically affected these parameters. Basolateral secretion of cytokine/chemokines by polarized Caco-2 cell monolayers was analysed by ELISA. RESULTS: Basophils loaded with OVA-specific IgE responded to OVA in a dose-dependent manner. OVA transported across polarized Caco-2 cell monolayers was found to trigger basolateral basophil activation. Microorganisms including lactobacilli and Escherichia coli increased transepithelial electrical resistance while promoting OVA passage capable to trigger basophil activation. Non-inflammatory levels of IL-8 and thymic stromal lymphopoietin were produced basolaterally by Caco-2 cells exposed to microorganisms. CONCLUSION: The complex model designed in here is adequate to learn about the consequence of the interaction between microorganisms and epithelial cells vis-a-vis the barrier function and antigen translocation, two parameters essential to mucosal homeostasis. It can further serve as a direct tool to search for microorganisms with anti-allergic and anti-inflammatory properties.