Fetal Intestinal Organ Culture Research Articles

Hydrocortisone-induced anti-inflammatory effects in immature human enterocytes depend on the timing of exposure.

The immature human gut has a propensity to exaggerated inflammatory responses that are thought to play a role in the pathogenesis of necrotizing enterocolitis (NEC). Prenatal exposure to corticosteroids has been reported to reduce the risk of NEC, while postnatal dexamethasone treatment is associated with adverse neurodevelopmental outcomes in preterm infants. The aim of this study was to investigate the direct role of hydrocortisone in gene expression patterns and inflammatory responses in immature human enterocytes. Time-dependent hydrocortisone effects in nontransformed primary human fetal intestinal epithelial cell line H4 were investigated by cDNA microarray. Fetal intestinal organ culture and cell culture experiments were conducted. Inflammatory responses were induced by stimulation with IL-1β and TNF-α with and without hydrocortisone. IL-8 and IL-6 expression and secretion were measured as functional readout. Here we report time-dependent hydrocortisone-induced changes in gene expression patterns detected by cDNA microarray. Hydrocortisone significantly attenuated IL-1β-induced inflammatory responses in the immature human gut when administered at the time of the proinflammatory insult: IL-1β-induced IL-8 and IL-6 secretion in the fetal ileum as well as H4 cells were significantly reduced. Hydrocortisone also inhibited IL-8 secretion in response to TNF-α. In contrast, TNF-α-induced IL-8 secretion was not reduced in cells treated with hydrocortisone for 48 h before stimulation. Our observations provide a physiological basis for understanding the differential clinical effects of corticosteroids in the immature human gut depending on the timing of treatment.

Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut.

Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Tumor necrosis factor (TNF)-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells (IECs) was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human IECs and directly modulates IEC innate immune gene expression.

Breast Milk-Transforming Growth Factor-β2 Specifically Attenuates IL-1β-Induced Inflammatory Responses in the Immature Human Intestine via an SMAD6- and ERK-Dependent Mechanism

Background: Breast milk is known to protect the infant against infectious and immuno-inflammatory diseases, but the mechanisms of this protection are poorly understood. Objectives: We hypothesized that transforming growth factor-β₂ (TGF-β₂), an immunoregulatory cytokine abundant in breast milk, may have a direct anti-inflammatory effect on immature human intestinal epithelial cells (IECs). Methods: Human fetal ileal organ culture, primary human fetal IECs, and the human fetal small intestinal epithelial cell line H4 were stimulated with interleukin 1β (IL-1β) with or without TGF-β₂. Pro-inflammatory cytokine secretion and mRNA expression were measured by ELISA and quantitative real-time polymerase chain reaction, respectively. Alterations in ERK signalling were detected from IECs by immunoblotting and in fetal ileal tissue culture by immunohistochemistry. SMAD6 knockdown was performed by transfecting the cells with SMAD6 siRNA. Results: TGF-β₂ significantly attenuated IL-1β-induced pro-inflammatory cytokine production in fetal intestinal organ culture and the cell culture models. In addition, TGF-β₂ reduced the IL-1β-induced IL-8 and IL-6 mRNA response in H4 cells. TGF-β₂ markedly inhibited IL-1β-induced phosphorylation of ERK, which was necessary for the cytokine response. The inhibitory effect of TGF-β₂ on IL-1β-induced cytokine production was completely abrogated by SMAD6 siRNA knockdown. Conclusions: TGF-β₂ attenuates IL-1β-induced pro-inflammatory cytokine production in immature human IECs by inhibiting ERK signalling. The anti-inflammatory effect of TGF-β₂ is dependent on SMAD6. Breast milk TGF-β₂ may provide the neonate with important immunoregulatory support. TGF-β₂ might provide a novel means to improve intestinal immunophysiology in premature neonates.

Adaptation of human enteric coronavirus to growth in cell lines.

Background: The existence of human enteric coronavirus (HEC) has been debated since its first description in stool by electron microscopy (EM) in 1975. Needed to resolve the issue is its cultivation in readily available cell lines.Objectives: To grow HEC in cell lines. To describe its characteristics and to differentiate it from other human and animal coronaviruses.Study design: Originally grown in human fetal intestinal organ culture, HEC was passed in J774 cells (a mouse macrophage cell line) and C6/36 cells (a mosquito cell line). Its cytopathic effect (CPE) and pattern of immunofluorescence were described. Its appearance was ascertained by negative staining and transmission EM. Its structural proteins were delineated by polyacrylamide gel electrophoresis (PAGE) and Western blotting (WB). The antigenic character of the virus was determined by immunofluorescence and WB. Agglutination with mouse erythrocytes was performed.Results: In J774 cells, HEC induced the formation of giant cells and small syncytia. Immunofluorescence in both J774 and C6/36 cells was limited to the cytoplasm. Studies with transmission EM revealed the virus to have the typical appearance of other coronaviruses, to be 80–120 nm in diameter, and to bud into cysternae of the endoplasmic reticulum. By PAGE and WB, its major protein has an average molecular weight (MW) of 41 kilodaltons (kDa). Two other proteins had MWs of 190 and 24 kDa. By immunofluorescence and WB, HEC is antigenically distinct from human coronaviruses 0C43 and 229E and mouse hepatitis virus (A59 strain). Preparations of HEC did not agglutinate mouse erythrocytes.Conclusion: We conclude that HEC is a human coronavirus that is antigenically unrelated to 0C43 and 229E viruses. Growth of HEC in readily available cell lines should aid in elucidating its role as a pathogen in human diarrheal illnesses.

Cyclosporin A enhances T cell-mediated induction of E-selectin.

In a fetal intestinal organ culture model of intestinal inflammation, activation of T cells by a lectin, superantigen or anti-CD3 antibodies induces E-selectin expression which peaks 6-10 h after activation and disappears by 24 h. We now show that addition of cyclosporin A (Cys A) increases the number of E-selectin-expressing endothelial cells and the intensity of expression, and it prolongs expression up to at least 60 h after stimulation. The dynamics of E-selectin expression on tumor necrosis factor activated human umbilical vein endothelial cell cultures is not affected by Cys A. Furthermore, other immunosuppressive agents FK506 and dexamethasone inhibit E-selectin expression, indicating that the enhancement observed in the presence of Cys A is not a consequence of general immunosuppression.

Behavior of fetal intestinal organ culture explanted onto a collagen substratum

A model of organ culture of 18 day old fetal rat intestine (Quaroni, 1985) was modified and characterized in the present work with the purpose of developing an in vitro model for the study of intestinal epithelial cell behaviour. Fragments of this intestine were kept in suspension culture for 7 days and then explanted onto collagen (type I) matrix. Within a day, the fragments became anchored to the substratum and a circular monolayer grew out to about 1 cm diameter. In the fragments, an outer layer of absorptive epithelial cells came to enclose a stroma, which was polarized into a loose (mesenchymal) and a dense portion. The dense portion contained a mixture of smooth muscle cells and primitive stem-type epithelial cells ('p-cells'). After explantation, at the contact point with the matrix, the epithelium broke up and the mesenchyme grew into the matrix and anchored the fragment. The epithelial edges now became continuous with the developing monolayer. Radioautography with tritiated thymidine indicated a constant cell renewal in epithelium and monolayer apparently from foci of p-cells, a reserve population of which was seen to be sequestered among the smooth muscle cells. Activated stem cells could differentiate into three mature epithelial phenotypes, each differentiation pathway apparently being determined by the type of underlying stroma. Immunohistochemistry using gold- and fluorescein-labeled monoclonal antibodies indicated that adult differentiation-specific markers (e.g. brush border enzymes) were present in the fragment epithelium but not in the monolayer cells. On the other hand, the monolayer cells could be induced to express some of these markers by contact with mesenchymal cells or by co-culturing with fibroblastic cell lines. Matrigel substratum mixed with collagen (type I) supported the appearance in monolayer of strands positive for amino-peptidase and lactase. The model thus appears to be suitable for the in vitro study of epithelial renewal and differentiation, and it has already provided some results in this respect.

Development of fetal rat intestine in organ and monolayer culture.

Maturation and differentiation of intestinal epithelial cells was demonstrated in segments of fetal rat small intestine, maintained for more than a month in suspension organ culture, by ultrastructural, biochemical, and immunological criteria. Over a 5-7 d period, fragments of fetal intestine evolved into globular structures covered with a single columnar epithelium ultrastructurally similar to suckling villus cells. Loose mesenchymal cells, cellular debris, and collagen were present inside the structures. After 6 d in culture, goblet cells, not present in the fetal intestine at day 18, were numerous and well developed. Intestinal endocrine cells were also observed. Immunofluorescence studies employing monoclonal antibodies specific for villus and crypt cells in vivo, and various enzyme assays, have demonstrated a level of differentiation and maturation of the cultured epithelial cells similar but not identical to that of suckling intestinal mucosa in vivo. Crypts and crypt cell markers were not observed in the the cultures. Addition of glucocorticoids to the culture medium resulted in the induction of sucrase-isomaltase but failed to promote most of the functional changes characteristic of the intestinal epithelium at weaning in vivo. Epithelial cells were identified in explants derived from the organ cultures by their specific expression of intestinal cytokeratin. Differentiation-specific markers, present in the epithelial cells in primary cultures, were lost upon selection and subculturing of pure epithelial cell populations. These results suggest a requirement for mesenchymal and/or extracellular matrix components in the maintenance of the differentiated state of the epithelial cells. The fetal intestinal organ cultures described here present significant advantages over traditional organ and monolayer culture techniques for the study of the cellular and molecular interactions involved in the development and differentiation of the intestinal epithelium.

In Vitro Cultivation in Human Fetal Intestinal Organ Culture of a Reovirus-Like Agent Associated with Nonbacterial Gastroenteritis in Infants and Children

Evidence was obtained that a reovirus-like agent associated with infantile gastroenteritis grew in organ cultures of human fetal intestine. Three serial passages of the agent in organ culture were successfully accomplished. Specific antigen was visualized in organ cultures by indirect immunofluorescence and appeared to be localized primarily in the epithelium that lined the intestinal villi. Immunofluorescence, with use of sections of infected organ cultures, was a sensitive method for serologic diagnosis of infection with the reovirus-like agent.

Ultrastructural study of viral replication in human fetal intestinal organ culture.

Human fetal intestinal organ cultures were employed to study the growth patterns of various viruses and resultant cytoarchitectural changes using electron microscopic techniques. Representative strains of adenovirus, adenovirusassociated virus, herpes simplex virus, poliovirus, and echovirus were chosen for the study. The intracellular localization of these viruses was demonstrated, and the occurrence and rate of cellular destruction were observed. The difficulty of visualizing smaller viruses represented was emphasized. The use of these techniques to detect certain fastidious viruses of the gastrointestinal tract was suggested, since human fetal intestinal organ culture provides differentiated cell types which may be important for the initiation and support of viral replication.

Detection and localization of viruses in human fetal intestinal organ cultures by immunofluorescence.

Viral antigens from viruses belonging to four different viral groups were detected directly in human fetal intestinal organ cultures by the application of immunofluorescent techniques. The time of appearance and the cellular localization of fluorescent-stainable antigen varied with the type of virus under investigation. After infection with adenovirus or with adeno-associated virus, fluorescent-stainable antigen was seen in the epithelial cells of the explants, though no light microscopic changes could be observed. In infection with herpes simplex virus and echovirus, fluorescence was noted in both the epithelium and the lamina propria, along with histological changes throughout the organ culture. These techniques offer promise for the investigation of possible viral agents implicated in gastrointestinal disease.

Biological Properties of Norwalk Agent of Acute Infectious Nonbacterial Gastroenteritis

Acute infectious nonbacterial gastroenteritis was induced in adult volunteers through three serial passages by oral administration of bacteria-free stool filtrates. This suggested that a replicating agent of subbacterial size was responsible for the observed disease. The biophysical properties of the agent, as assayed in volunteers, were consistent with those of a small virus, most closely related to the parvovirus group among known animal viruses. The agent appeared to have a diameter less than 66 nm and likely less than 36 nm. It appeared to lack a lipid containing envelope and was acid stable. It was stable to heating at 60° for 30 min. The agent appeared to be relatively host specific for man and conferred at least short-term immunity. Because of the high frequency of disease induced in unselected volunteers, widespread natural immunity to this agent may be absent or perhaps incomplete. Preliminary evidence suggested that the Norwalk agent replicated in an in vitro system, human fetal intestinal organ culture.

Establishment of human fetal intestinal organ cultures for growth of viruses.

The advent of modern techniques for the laboratory cultivation of whole organ explants has led to the establishment of cultures of a wide variety of organs from a number of different hosts [1]. The small intestine, however, has proven to be a notable exception. Previous attempts at intestinal and other organ cultures have utilized a variety of techniques [2-6]. Enders and his co-workers reported survival of fragments of human embryonic intestine for at least 17 days in a modified Maitland culture system [7], but viable human intestinal organ cultures, lasting more than 24 hr, have not been described. Stimulated by recent experience in the detection of occult respiratory viruses through the use of tracheal organ cultures [8, 9], we have established small intestinal organ cultures from human fetal material, as well as from chicken and rabbit embryos, and have maintained these cultures for up to 21 days. Hopefully, these organ cultures may offer a sensitive system for the replication and detection of heretofore occult viral