Objective The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualised medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (ie, barrier function and host–microbial interactions). Design We created a large panel of human gastrointestinal epithelial cell lines (n=65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays. Results We obtained epithelial lines from all accessible tissue sites within 2 weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3–1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarised monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli . Conclusions This culture system will facilitate the study of interindividual, functional studies of human intestinal epithelial cells, including host–microbial interactions.

https://gut.bmj.com/content/gutjnl/early/2014/07/09/gutjnl-2013-306651.full.pdf

Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays

The gut-associated lymphoid tissue (GALT) consists of isolated or aggregated lymphoid follicles forming Peyer's patches (PPs). By their ability to transport luminal antigens and bacteria, PPs can be considered as the immune sensors of the intestine. PPs functions like induction of immune tolerance or defense against pathogens result from the complex interplay between immune cells located in the lymphoid follicles and the follicle-associated epithelium. This crosstalk seems to be regulated by pathogen recognition receptors, especially Nod2. Although TLR exerts a limited role in PP homeotasis, Nod2 regulates the number, size, and T-cell composition of PPs, in response to the gut flora. In turn, CD4

https://downloads.hindawi.com/journals/iji/2010/823710.pdf

Peyer's Patches: The Immune Sensors of the Intestine

We have investigated 677 Shiga toxin-producing Escherichia coli (STEC) strains from humans to determine their serotypes, virulence genes, and clinical signs in patients. Six different Shiga toxin types (1, 1c, 2, 2c, 2d, and 2e) were distributed in the STEC strains. Intimin (eae) genes were present in 62.6% of the strains and subtyped into intimins alpha1, beta1, gamma1, epsilon, theta, and eta. Shiga toxin types 1c and 2d were present only in eae-negative STEC strains, and type 2 was significantly (P < 0.001) more frequent in eae-positive STEC strains. Enterohemorrhagic E. coli hemolysin was associated with 96.2% of the eae-positive strains and with 65.2% of the eae-negative strains. Clinical signs in the patients were abdominal pain (8.7%), nonbloody diarrhea (59.2%), bloody diarrhea (14.3%), and hemolytic-uremic syndrome (HUS) (3.5%), and 14.3% of the patients had no signs of gastrointestinal disease or HUS. Infections with eae-positive STEC were significantly (P < 0.001) more frequent in children under 6 years of age than in other age groups, whereas eae-negative STEC infections dominated in adults. The STEC strains were grouped into 74 O:H types by serotyping and by PCR typing of the flagellar (fliC) genes in 221 nonmotile STEC strains. Eleven serotypes (O157:[H7], O26:[H11], O103:H2, O91:[H14], O111:[H8], O145:[H28], O128:H2, O113:[H4], O146:H21, O118:H16, and O76:[H19]) accounted for 69% of all STEC strains. We identified 41 STEC strains belonging to 31 serotypes which had not previously been described as human STEC. Twenty-six of these were positive for intimins alpha1 (one serotype), beta1 (eight serotypes), epsilon (two serotypes), and eta (three serotypes). Our study indicates that different types of STEC strains predominate in infant and adult patients and that new types of STEC strains are present among human isolates.

Characterization of Shiga Toxin-Producing Escherichia coli Strains Isolated from Human Patients in Germany over a 3-Year Period

Mucus provides protective functions in the gastrointestinal tract and plays an important role in the adhesion of microorganisms to host surfaces. Mucin glycoproteins polymerize, forming a framework to which certain microbial populations can adhere, including probiotic Lactobacillus species. Numerous mechanisms for adhesion to mucus have been discovered in lactobacilli, including partially characterized mucus binding proteins. These mechanisms vary in importance with the in vitro models studied, which could significantly affect the perceived probiotic potential of the organisms. Understanding the nature of mucus-microbe interactions could be the key to elucidating the mechanisms of probiotic adhesion within the host.

https://www.mdpi.com/2072-6643/3/5/613/pdf

Lactobacillus Adhesion to Mucus

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome worldwide. Conventional antimicrobials trigger an SOS response in EHEC that promotes the release of the potent Shiga toxin that is responsible for much of the morbidity and mortality associated with EHEC infection. Cattle are a natural reservoir of EHEC, and approximately 75% of EHEC outbreaks are linked to the consumption of contaminated bovine-derived products. This review will discuss how EHEC causes disease in humans but is asymptomatic in adult ruminants. It will also analyze factors utilized by EHEC as it travels through the bovine gastrointestinal tract that allow for its survival through the acidic environment of the distal stomachs, and for its ultimate colonization in the recto-anal junction. Understanding the factors crucial for EHEC survival and colonization in cattle will aid in the development of alternative strategies to prevent EHEC shedding into the environment and consequent human infection.

/pdf/enterohemorrhagic-e-coli-ehec-pathogenesis-5xcwveepc5.pdf

Enterohemorrhagic E. coli (EHEC) pathogenesis

BACKGROUND—Enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide, and infections, particularly with serogroup O157:H7, are associated with consumption of a variety of food and water vehicles, particularly food of bovine origin. EHEC cause acute gastroenteritis, bloody diarrhoea, and haemorrhagic colitis; up to 10% of cases develop severe complications, including the haemolytic uraemic syndrome, with a 5% case fatality. A virulence characteristic of enteropathogenic E coli, the attaching/effacing lesion, is considered to be important in EHEC. However, although EHEC produce this lesion on cultured human cells, this has not been demonstrated on human intestinal mucosal surfaces. In addition, the initial site(s) of colonisation of EHEC in humans is not known.
AIMS—To assess the association of EHEC O157:H7 with paediatric and bovine intestine using in vitro organ culture and determine if attaching/effacing lesions occur.
METHODS—Ultrastructural analysis of in vitro intestinal organ cultures of human small and large intestine was used to investigate adhesion of O157:H7 EHEC to intestinal surfaces. Bovine intestinal organ culture was used to examine the pathology produced by the same EHEC strain in cattle.
RESULTS—The study showed that EHEC O157:H7 adhered to human intestinal mucosa. Binding and attaching/effacing lesion formation of O157:H7 in humans was restricted to follicle associated epithelium of Peyer's patches. The same strain caused attaching/effacing lesions on bovine mucosa.
CONCLUSIONS—O157:H7 targets follicle associated epithelium in humans where it causes attaching/effacing lesions. The same human isolate can cause attaching/effacing lesions in cattle, indicating that similar pathogenic mechanisms operate across human and bovine species


Keywords: Escherichia coli; enterohaemorrhagic E coli O157:H7; Peyer's patches; attaching/effacing lesion

https://gut.bmj.com/content/gutjnl/47/3/377.full.pdf

Enterohaemorrhagic Escherichia coli O157:H7 target Peyer's patches in humans and cause attaching/effacing lesions in both human and bovine intestine.

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Enterohaemorrhagic Escherichia coli O157:H7 target Peyer's patches in humans and cause attaching/effacing lesions in both human and bovine intestine.