Human Intestinal Organ Culture Research Articles

Antigen-Presenting Human γδ T Cells Promote Intestinal CD4+ T Cell Expression of IL-22 and Mucosal Release of Calprotectin.

The cytokine IL-22 plays a critical role in mucosal barrier defense, but the mechanisms that promote IL-22 expression in the human intestine remain poorly understood. As human microbe–responsive Vγ9/Vδ2 T cells are abundant in the gut and recognize microbiota-associated metabolites, we assessed their potential to induce IL-22 expression by intestinal CD4+ T cells. Vγ9/Vδ2 T cells with characteristics of APCs were generated from human blood and intestinal organ cultures, then cocultured with naive and memory CD4+ T cells obtained from human blood or the colon. The potency of blood and intestinal γδ T-APCs was compared with that of monocytes and dendritic cells, by assessing CD4+ T cell phenotypes and proliferation as well as cytokine and transcription factor profiles. Vγ9/Vδ2 T cells in human blood, colon, and terminal ileum acquired APC functions upon microbial activation in the presence of microenvironmental signals including IL-15, and were capable of polarizing both blood and colonic CD4+ T cells toward distinct effector fates. Unlike monocytes or dendritic cells, gut-homing γδ T-APCs employed an IL-6 independent mechanism to stimulate CD4+ T cell expression of IL-22 without upregulating IL-17. In human intestinal organ cultures, microbial activation of Vγ9/Vδ2 T cells promoted mucosal secretion of IL-22 and ICOSL/TNF-α–dependent release of the IL-22 inducible antimicrobial protein calprotectin without modulating IL-17 expression. In conclusion, human γδ T-APCs stimulate CD4+ T cell responses distinct from those induced by myeloid APCs to promote local barrier defense via mucosal release of IL-22 and calprotectin. Targeting of γδ T-APC functions may lead to the development of novel gut-directed immunotherapies and vaccines.

Human milk oligosaccharides and galactosyloligosaccharides attenuate inflammation in human intestine

The immature intestinal mucosa is hyperresponsive to exogenous and endogenous inflammatory insults. Human milk provides immune protection to infants, and its oligosaccharides (HMOS) are putative immunomodulators. Of these, galactosyloligosaccharides (GOS) are high in colostrum, but not mature milk. The objective is to determine the ability of GOS to directly attenuate inflammation, and potential mechanisms investigated. Human models of immature and mature intestine were used to investigate the ability of a mixture of HMOS or synthetic GOS to attenuate inflammation induced by proinflammatory stimuli and enteric pathogens. These results were confirmed in the ex vivo immature human intestinal organ cultures. The ability of TNFα and of pathogens to elicit pro‐inflammatory neutrophil chemoattractants IL‐8, MIP‐3α and MCP1 and their mRNA levels was significantly attenuated by HMOS and GOS. TNFα‐induced nuclear translocation of NF‐κB was mitigated by HMOS and GOS. Consistent with previous indications of bioactivity of galactosyllactose from human colostrum, trimeric galactosyloligosaccharides interact with cell receptors to inhibit inflammatory response. HMOS and GOS suppress NF‐κB activation, thereby directly attenuating TNFα‐ and pathogen‐induced inflammation in human intestine. This provides a novel mechanism whereby specific galactosyloligosaccharides found in human milk may prove a novel therapeutic agent against gut inflammation.

Initiation of an inflammatory response in resident intestinal lamina propria cells -use of a human organ culture model.

Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL1B, IL6, IL8, IL23A, TNFA, CXCL2, and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. Furthermore, genes never been previously associated with intestinal inflammation were identified using this model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease.

Intimin-mediated tissue specificity in enteropathogenic Escherichia coli interaction with human intestinal organ cultures.

The hallmark of enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) adhesion to cultured human host cells is intimate attachment and the formation of attaching and effacing (A/E) lesions. Recently, EHEC O157:H7 was shown to induce A/E lesions on human intestinal explants. Unlike EPEC, which colonized the small intestine, EHEC adhesion was restricted to follicle-associated epithelium (FAE) of ileal Peyer's patches. This study tested the hypothesis that the bacterial adhesin intimin contributes to tissue specificity. Complementing the eae gene mutation in CVD206 (derived from EPEC strain E2348/69) with EPEC eaealpha (encoding intimin-alpha) restored the ability to colonize small intestinal mucosa like the parent strain. In contrast, complementing with EHEC eaegamma (encoding intimin-gamma) resulted in the strain adhering and inducing A/E lesion on Peyer's patches, similar to EHEC. An intimin-gamma-positive O55:H7 EPEC also targeted FAE. Thus, intimin contributes to the tissue specificity of A/E lesion-forming microbial pathogens.

Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro.

Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 (eae deficient), CVD206(pCVD438) (eae-complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a TnphoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206 (pCVD438) (both Int+ BFP+) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int+ BFP-) adhered and caused A/E lesions although bacteria adhered in "flat," two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int- BFP+) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.

Both apolipoproteins B-48 and B-100 are synthesized and secreted by the human intestine

Apolipoprotein B (apoB), an apolipoprotein associated with very low density lipoproteins and the atherogenic low density lipoproteins (LDL), directs the metabolism of lipoprotein particles in plasma by interacting with the LDL receptor. Utilizing human intestinal biopsy organ cultures, we have studied the synthesis of intestinal apoB in man. Intestinal organ cultures from normal adults (n = 6) were incubated in the presence of protease inhibitors in media supplemented with [35S]methionine. Media from these cultures were evaluated by sequential NaDodSO4 polyacrylamide gel electrophoresis, radioautography, and Western blot analyses, and intestinal biopsies were studied using immunohistochemistry. The relative abundance of apoB-100 and apoB-48 mRNA was assessed using reverse transcriptase-polymerase chain reaction followed by primer extension. Although apoB-48 was the principal isoprotein that was newly synthesized by intestinal organ cultures, apoB-100 was also synthesized and secreted by human intestinal organ cultures with 16 +/- 3% of the intestinal apoB mRNA coding for apoB-100. These results establish that apoB-100 is produced by the human intestine. The synthesis of the atherogenic apoB-100 by the intestine has pathophysiologic implications for the development of diet-induced atherosclerosis.

Correlation of cord bilirubin levels with hyperbilirubinaemia in ABO incompatibility.

We studied 91 offspring of ABO incompatible preganacies and 30 controls resulting from O--O pregnancies to test whether cord bilirubin levels could be used to predict the severity of hyperbilirubinaemia in ABO incompatibility. Blood group, direct Coombs's test, and serum bilirubin estimations were carried out on cord blood, and bilirubin estimations at 12, 24, 36, and 48 hours of life.

Further studies on human enteric coronaviruses

SummaryComparisons were made between human enteric coronaviruses and the enteric coronaviruses of pigs and calves by negative staining.Examination of human intestinal organ culture fluids at various time intervals after inoculation with the human enteric coronavirus showed increasing numbers of particles in the fluids.Thin sections of the columnar epithelial cells of these explants showed a number of features consistent with the replication of known human and animal coronaviruses. Virus particles found in thin sections had a mean diameter of 68 nm. In addition, a structure was found in thin sections which has not been described previously. This structure may represent the viral nucleocapsid.

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