Fetal Intestinal Tissue Research Articles

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids.

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

The Amoebicidal Effect of Ergosterol Peroxide Isolated from Pleurotus ostreatus.

Dysentery is an inflammation of the intestine caused by the protozoan parasite Entamoeba histolytica and is a recurrent health problem affecting millions of people worldwide. Because of the magnitude of this disease, finding novel strategies for treatment that does not affect human cells is necessary. Ergosterol peroxide is a sterol particularly known as a major cytotoxic agent with a wide spectrum of biological activities produced by edible and medicinal mushrooms. The aim of this report is to evaluate the amoebicidal activity of ergosterol peroxide (5α, 8α-epidioxy-22E-ergosta-6,22-dien-3β-ol isolated from 5α, 8α-epidioxy-22E-ergosta-6,22-dien-3β-ol) (Jacq.) P. Kumm. f. sp. Florida. Our results show that ergosterol peroxide produced a strong cytotoxic effect against amoebic growth. The inhibitory concentration IC50 of ergosterol peroxide was evaluated. The interaction between E. histolytica and ergosterol peroxide in vitro resulted in strong amoebicidal activity (IC50 = 4.23 nM) that may be due to the oxidatory effect on the parasitic membrane. We also tested selective toxicity of ergosterol peroxide using a cell line CCL-241, a human epithelial cell line isolated from normal human fetal intestinal tissue. To the best of our knowledge, this is the first report on the cytotoxicity of ergosterol peroxide against E. histolytica, which uncovers a new biological property of the lipidic compound isolated from Pleurotus ostreatus (Jacq.) P. Kumm. f. sp. Florida.

Anisotropic growth shapes intestinal tissues during embryogenesis

Embryogenesis offers a real laboratory for pattern formation, buckling, and postbuckling induced by growth of soft tissues. Each part of our body is structured in multiple adjacent layers: the skin, the brain, and the interior of organs. Each layer has a complex biological composition presenting different elasticity. Generated during fetal life, these layers will experience growth and remodeling in the early postfertilization stages. Here, we focus on a herringbone pattern occurring in fetal intestinal tissues. Common to many mammalians, this instability is a precursor of the villi, finger-like projections into the lumen. For avians (chicks' and turkeys' embryos), it has been shown that, a few days after fertilization, the mucosal epithelium of the duodenum is smooth, and then folds emerge, which present 2 d later a pronounced zigzag instability. Many debates and biological studies are devoted to this specific morphology, which regulates the cell renewal in the intestine. After reviewing experimental results about duodenum morphogenesis, we show that a model based on simplified hypothesis for the growth of the mesenchyme can explain buckling and postbuckling instabilities. Being completely analytical, it is based on biaxial compressive stresses due to differential growth between layers and it predicts quantitatively the morphological changes. The growth anisotropy increasing with time, the competition between folds and zigzags, is proved to occur as a secondary instability. The model is compared with available experimental data on chick's duodenum and can be applied to other intestinal tissues, the zigzag being a common and spectacular microstructural pattern of intestine embryogenesis.

TRANSLATIONAL IMPACT

Enterohemorrhagic Escherichia coli (EHEC) is an emerging zoonotic pathogen that causes acute gastroenteritis and hemorrhagic colitis in humans. It also causes disease in newborn calves and asymptomatically colonizes the gut mucosa of adult bovines, therefore constituting the main reservoir for food and environmental contamination. The hallmark gut lesion induced by EHEC involves bacterial attachment to the gut epithelium and effacing of gut microvilli, but innate immune responses and inflammation are conspicuously limited. Given that EHEC is strictly a human and calf pathogen that does not infect mice, it has been a challenge to develop simple models that permit detailed investigation of virulence mechanisms. Tissue tropism cannot be mimicked in vitro because EHEC is neither species- nor cell-type-specific in culture. Although human in vitro organ cultures have been used, this model has a short lifespan, does not accurately mimic normal gut architecture and lacks an intact immune system. Therefore, a better model is needed to study the molecular bases for the human and calf specificity of EHEC, its tissue tropism and the innate immune response induced upon infection.To address this need, the authors developed an in vivo model of EHEC infection by xenotransplantation of human or bovine fetal intestinal tissue under the skin of SCID mice (which do not reject the foreign tissue owing to lack of an adaptive immune system). Xenografts of human and bovine tissue originally isolated from different gut regions develop into mature tissues and faithfully mimic the tissue of human and calf intestine. Infection of xenografts with EHEC induces the formation of typical attaching and effacing lesions and tissue damage resembling hemorrhagic colitis. By contrast, xenografts infected with an EHEC mutant deficient in the type III secretion system (a major EHEC virulence mechanism) remained undamaged. Notably, EHEC does not attach to or damage the epithelium of small intestinal tissue.This xenograft model of EHEC infection faithfully recapitulates several aspects of the natural human and calf disease, including host and tissue tropism, typical histopathology, and attenuation of innate immune responses. Therefore, this relatively simple and economical xenograft model should be effective for resolving molecular mechanisms of host and tissue tropism, as well as the immunomodulatory effects of EHEC in intact tissue. A challenging step to further developing this model would be to connect the xenografted segments within the intestines of mice in order to establish a system that is fully reconstituted with normal microflora as well as the human or bovine immune systems.

Interleukin-8/CXCL8 forms an autocrine loop in fetal intestinal mucosa.

IL-8/CXC ligand (CXCL) 8 is ingested in high concentrations by the human fetus/neonate with amniotic fluid and human milk, and is also produced constitutively by intestinal epithelial cells (IEC). We have shown that recombinant human IL-8/CXCL8 (rhIL-8/CXCL8) protects cultured IEC against tumor necrosis factor (TNF)-alpha and cycloheximide-induced cytotoxicity. In view of its constitutive production, we hypothesized that IL-8/CXCL8 might play an autocrine role in fetal enterocyte maintenance. In this study, we measured IL-8/CXCL8 mRNA concentrations in fetal intestine (11-22 wk gestation), sought the presence of the protein by immunohistochemistry in fetal stomach and intestine (9-24 wk), measured IL-8/CXCL8 in neonatal gastric secretions, and studied constitutive and stimulated IL-8/CXCL8 expression in cultured IEC. We found that IL-8/CXCL8 is consistently transcribed and expressed in fetal intestinal tissue, in a developmentally regulated inverse relationship with gestational maturation. The cognate receptors for IL-8/CXCL8 are also expressed abundantly in the fetal intestine, and, therefore, we sought to determine whether the expressed IL-8/CXCL8 would complete an autocrine loop. Neutralization of IL-8/CXCL8 resulted in increased cell death in cultured IEC in the presence of TNF-alpha. This effect is specifically mediated through the CXCR2 receptors. We speculate that IL-8/CXCL8 secretion during cytotoxic stress reflects a cellular self-defense mechanism.

Human placenta and fetal membranes contain peptide YY1-36 and peptide YY3-36.

Extracts of human term amniotic, placental, and chorion/decidua tissue contained, respectively, 4.36 +/- 2.79 (pmol/g wet wt; mean +/- S.E.M.: n = 5). 2.78 +/- 0.5 (n = 5) and 0.68 +/- 0.68 (n = 5) peptide YY (PYY)-like immunoreactivity. Using a specific PYY antiserum, gel filtration chromatography and reverse-phase high performance liquid chromatography (HLPC), amniotic, placental and fetal intestinal tissue extracts were demonstrated to contain PYY-like immunoreactivity consisting of equal amounts of PYY1-36 and PYY3-36. The presence of pancreatic polypeptide was not detected in any of the extracts. Positive immunohistochemical staining for PYY was seen in extravillous trophoblasts in the decidual septa and fetal membranes, the syncytiotrophoblast and cytotrophoblasts, amniotic epithelial cells and in maternal decidual stromal cells. Positive staining for PYY was found at the earliest date examined (9.5 weeks) and remained present throughout pregnancy to term. PYY1-36 and PYY3-36 may play important roles in human pregnancy, acting via endocrine or paracrine mechanisms.

Failure of short-term luminal IGF-I to protect against atrophy in a model of fetal esophageal atresia

Short-term luminal infusion in utero (3 days) of insulin-like growth factor I (IGF-I) failed to protect the fetal small intestine against atrophy induced by ablation of swallowing. Human recombinant IGF-I (or vehicle) was infused into the duodenum of fetal sheep at 125 days' gestation for 3 days (day 1, 0.025 mg; day 2, 0.25 mg; day 3, 2.5 mg). Fetal swallowing was prevented by esophageal ligation, and a carotid catheter was implanted for blood sampling. There were no changes in body growth or in major organ growth. Small intestinal (SI) weight (corrected for body weight) was significantly lower for IGF-I treated fetuses. Villus height decreased significantly in proximal regions. Villus enterocyte cellularity was reduced significantly in the proximal regions. The percentage of crypt cells labeled with a 4-hour pulse of tritiated thymidine (as assessed by autoradiography) decreased significantly in the proximal SI only, from 16.14% (1.06% SEM) to 13.28% (1.05% SEM) (P < .05). Plasma levels of IGF-I increased in the treated fetuses by an average of 76%. IGF-I immunoreactivity was detected in the apical endocytic complex of enterocytes from proximal SI. This study shows that wasting of fetal intestinal tissues in the absence of enteral input cannot be prevented by IGF-I delivered luminally.

HIV infection of human fetal intestinal explant cultures induces epithelial cell proliferation

The concept that HIV infection per se alters small intestinal mucosal structure and function (HIV enteropathy) remains controversial and in this study we report in vitro experiments designed to elucidate the matter. Twenty pairs of human fetal intestinal tissue explants were maintained in vitro for up to 14 days; one explant of each pair was incubated and infected with HIV, and the other served as a matched uninfected control. At various times after infection, explant culture fluid and tissue were removed, p24 concentration was measured and tissue formalin fixed. Explant tissue was embedded in paraffin wax and sections stained by an immunoperoxidase method directed against proliferating cell nuclear antigen (PCNA). The percentage of proliferating crypt and villous epithelial cells, stained by PCNA, was calculated in paired samples. The difference between the percentage for paired samples was designated delta crypt proliferation (delta CP) and delta villous proliferation (delta VP), respectively. Epithelial cell proliferation was deemed to be enhanced if the percentage of PCNA-stained cells was greater in the HIV-infected than in the control tissue. Explant culture fluid from tissue exposed to HIV showed a progressive rise in p24 antigen (Ag) level, indicating HIV infection of these explants. Fifteen pairs of explants showed progressively positive delta CP with time (P < 0.01) indicating crypt hyperplasia and all 20 pairs of explants showed positive delta VP, indicating hyperplasia of villous epithelial cells. This study provides direct evidence that HIV stimulates epithelial cell proliferation in intestinal mucosa. HIV-infected human intestinal explants provide a model of crypt hyperplastic villous atrophy previously described as HIV enteropathy and detected in clinical biopsy specimens from HIV-infected patients.

Specific expression of unusual GM2 ganglioside with Hanganutziu-Deicher antigen activity on human colon cancers.

This paper reports the presence of GM2 ganglioside containing N-glycolylneuraminic acid (NeuGc) in human colon cancer tissues. GM2(NeuGc) was detected by two-dimensional thin layer chromatography (2d-TLC)/enzyme-immunostaining using affinity-purified chicken antibody against GM3(NeuGc) and horseradish peroxidase-conjugated rabbit anti-chicken IgG antibody. Like usual GM2 ganglioside containing N-acetylneuraminic acid (NeuAc) isolated from Tay-Sachs brain, GM2(NeuGc) in colon cancer could be converted into GM3(NeuGc) by human kidney beta-N-acetylhexosaminidase A in the presence of a GM2-specific activator protein isolated from guinea pig kidney. Three of 7 specimens of Hanganutziu-Deicher (HD) antigen-positive human colon cancer tissues so far examined expressed this unique ganglioside. In order to detect and determine specifically GM2(NeuGc) on human colon cancers, specific antibody against GM2 (NeuGc) has been prepared by immunizing chickens. By a sensitive TLC/immunostaining method using the antibody, the amounts of the antigen were determined to be 0.3-3% of total lipid-bound sialic acid. NeuGc-containing gangliosides were also detected in meconium and fetal intestinal tissues. Three species of antigenic gangliosides in pooled meconium were tentatively identified as GM3(NeuGc), sialylparagloboside and sialylhexaosylceramide on the basis of their migration positions on 2d-TLC and the results of endo-beta-galactosidase treatment. GM3(NeuGc) was the sole HD-active ganglioside in fetal intestinal tissue from one of 3 individuals tested; the other two showed no HD-active ganglioside at all. GM2(NeuGc), however, could not be detected in either meconium or fetal tissues so far examined, suggesting that this unique ganglioside is a tumor-specific antigen, at least for human intestinal tissues.

231 EXPLANT CULTURE OF HUMAN FETAL ESOPHAGUS, STOMACH AND COLON

We recently reported the successful maintenance of human fetal intestinal tissue for 9 days in serum-free organ culture (Ménard and Arsenault, Gastroenterology, in press). This investigation was undertaken in order to evaluate the possibility to extent to other fetal digestive tract tissues this serum-free organ culture technique. Explants of fetal esophagus, stomach and colon (10–15 weeks gestation) were maintained in serum-free Leibovitz L-15 medium at 37°C in 95% air - 5% CO2. As determined by light and electron microscopy the overall architecture of esophagus, stomach and colon explants was maintained for 9, 21 and 15 days, respectively. Furthermore, the epithelium of the stomach continued to differentiate. The incorporation of 3H-thymidine and 3H-leucine continued during the culture period, reflecting a sustained synthesis of DNA and proteins. On the other hand, the incorporation of 3H-glucosamine increased during the culture, pointing out to the possibility of an accelerated glycoprotein synthesis. As determined by radioautography, the proliferative cells were detected in the basal layer of the ciliated columnar epithelium of esophagus, at all levels of the stomach epithelium and in the inter-villus area of the colon. These observations clearly established that the entire human fetal digestive tract can be maintained in the same complete controlled environment. Therefore, this explant culture technique should be a useful tool for the study of the development of human fetal digestive tract.

230 CELL PROLIFERATION IN HUMAN FETAL SMALL INTESTINE

Cellular kinetic data are not available for human fetal intestinal tissue. Therefore an investigation was undertaken to establish the tritiated thymidine (3H-TdR) incorporation into DNA and the labelling indices (LI) in the epithelium (E), mesenchymal (ME) and the muscular (M) layers between 8 and 15 weeks gestation. Explants of small intestine were cultured in serum-free Leibovitz L-15 medium at 37°C in 95% air-5% CO2. The LI were established on radioautographs of explants cultured for 2 h in presence of 2 μCi of 3H-TdR per ml of culture medium. The incorporation of 3H-TdR into DNA was evaluated after a 6 h period and results expressed as DPM/μg DNA. Between 8 and 10 weeks' gestation, the LI were 28, 14 and 9% for respectively the E, ME and M layers. Between 10 and 12 weeks there was a decrease of the LI which remained more or less constant between 12 and 15 weeks (15, 9 and 6%). Labelled epithelial cells were noted at all levels of the epithelium prior to or at the beginning of villus formation and later on were observed only in the intervillous area. The addition of hydrocortisone (50 ng/ml) to the culture medium during 5 days induced a significant increase (98%) of 3H-TdR incorporation into DNA. This overall increase of DNA synthesis was reflected at the epithelial cell level where the LI increased by 57%. These experiments provide some basic data on cell proliferation in fetal intestine and indicate that explant culture technique should be useful for the study of the regulators of cell proliferation during the development of human intestine.

In utero effects of 1,2-dimethyl hydrazine on adenosine and guanosine 3′,5′-cyclic monophosphate content in rat fetal intestine

Sperm positive 12–14-day pregnant Holtzman rats were exposed to a single subcutaneous injection (20 mg/kg body wt) of 1,2-dimethylhydrazine (DMH). Two days post-exposure, the fetal intestinal tissues were examined for their content and metabolic activities for cyclic 3′,5′-adenosine monophosphate (cAMP) and cyclic 3′,5′-guanosine monophosphate (cGMP). The in utero exposure to the carcinogen resulted in lowering the intracellular content of cAMP and increasing cGMP. The decreased levels of cAMP may be accounted for the finding of a corresponding increase in its breakdown by its phosphodiesterases; however, associated with the increase in cGMP was correspondingly an increase in its phosphodiesterases, suggesting the activities for synthesis of this cyclic nucleotide may be the major factor for its elevated concentration. These observations indicate that DMH may cross the placental barrier and can effect the intracellular cyclic nucleotide concentrations in the fetal tissues as well as acting as a colorectal carcinogen in the adult rat. The changes in the cyclic nucleotide levels were toward the direction expected for an increased cell proliferation; consequently, further investigations are suggested to determine whether a hyperproliferative state is induced by the DMH in the already rapidly dividing fetal intestinal tissue.

Development of jejunoileal differences of activity of lactase, sucrase and acid beta-galactosidase in isografts of fetal rat intestine.

Jejunal and ileal segments from preterm rat fetuses were implanted under the kidney capsula of adult rats. Sucrase, lactase and acid beta-galactosidase activities were determined in the isografts at different times after implantation, and in corresponding segments developing in situ. Whereas fetal intestine contains considerable activity of acid beta-galactosidase and lactase, no sucrase activity is detectable. Similarly -- as in situ -- 4 weeks after the implantation the jejunal segment exhibited higher activity of sucrase and lactase than the ileal segment. Acid beta-galactosidase was more active in ileal than in jejunal segments -- both growing in situ as well as isografts. Experiments have thus demonstrated that the expression of the jejunoileal gradient of activity of the 3 enzymes studied does not depend on direct contact with food or gastric, pancreatic and biliary juices. This gives validity to the suggestion that the gradient may already be programmed in fetal intestinal tissue, but other factors active in situ might be responsible for its magnitude.

Immunofluorescence Studies on the Pathogenesis of Intestinal Chlamydial Infections in Calves

Newborn calves were exposed orally to the chlamydial agent of bovine polyarthritis. The chlamydial infection in the gastrointestinal tract was traced by reisolation of the agent and by fluorescent-antibody techniques. Absorption of the fluorescein isothiocyanate (FITC)-labeled antiserum with bovine fetal intestinal tissue powder eliminated effectively the nonspecific fluorescence of eosinophilic granules in intestinal tissue sections. Cells of the eosinophilic series were observed in great numbers in the gastrointestinal tract of inoculated and normal calves. Although chlamydial agents could be reisolated from mucosal scrapings of abomasum and duodenum for 5 days after inoculation, specific fluorescence was not observed in these gastrointestinal portions. As the chlamydial infection progressed, it localized in the mucosal epithelial cells of the jejunum and ileum. Fluorescing chlamydial inclusions were observed most consistently in the cytoplasm of mucosal epithelial cells on the tips of the jejunal and ileal villi. The inclusions were located between the nucleus and the free border of the epithelial cells. In the deeper parts of the villi, the inclusions in the epithelial cells were situated frequently between the nucleus and the basement membrane of the mucosal lamina propria. In calves examined 7 days after inoculation, fluorescing chlamydial inclusions were seen in the cells of the crypts and the mucosal lamina propria of the lower portions of the small intestine. Chlamydial infection of cells in the intestinal interstitium reflected a process of systemic invasion.