Anders Edebo

Mucosal FOXP3-expressing CD4+ CD25high regulatory T cells in Helicobacter pylori-infected patients

ABSTRACT Helicobacter pylori chronically colonizes the stomach and duodenum and causes peptic ulcers or gastric adenocarcinoma in 10 to 20% of infected individuals. We hypothesize that the inability of patients to clear H. pylori infections is a consequence of active suppression of the immune response. Here we show that H. pylori-infected individuals have increased frequencies of CD4+ CD25high T cells in both the stomach and duodenal mucosa compared to uninfected controls. These cells have the phenotype of regulatory T cells, as they express FOXP3, a key gene for the development and function of regulatory T cells, as well as high levels of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) protein. In contrast, mucosal CD4+ CD25low and CD4+ CD25− cells express little FOXP3 mRNA and low levels of the CTLA-4 protein. Mucosal CD4+ CD25high T cells are present in individuals with asymptomatic H. pylori infections as well as in duodenal ulcer patients. The frequencies of CD4+ CD25high cells are also increased in the stomachs of H. pylori-infected patients with gastric adenocarcinoma, particularly in cancer-affected tissues. These findings suggest that regulatory T cells may suppress mucosal immune responses and thereby contribute to the persistence of H. pylori infections.

Helicobacter pylori-Specific CD4+ T Cells Home to and Accumulate in the Human Helicobacter pylori-Infected Gastric Mucosa

ABSTRACT Helicobacter pylori infects the stomach and duodenal mucosa. T cells are important components of the H. pylori-induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated from H. pylori-infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity to H. pylori antigens. Higher numbers of CD4+ but similar numbers of CD8+ lamina propria T cells were isolated from stomach biopsies from H. pylori-positive compared to H. pylori-negative individuals. CD4+ T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4+ T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4+ T cells with similar receptor expression were isolated from the duodenum of H. pylori-positive and H. pylori-negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach, H. pylori-specific T-cell responses were detected in stomach CD4+ T cells from H. pylori-positive but not H. pylori-negative individuals. Duodenal T cells from only a few individuals responded to H. pylori stimulation, and the responsiveness was not restricted to H. pylori-positive individuals, suggesting limited H. pylori specificity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest that H. pylori-specific CD4+ T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment.

Increased frequency of activated T-cells in the Helicobacter pylori-infected antrum and duodenum

Helicobacter pylori colonize the human stomach and duodenum. The infection has been shown to induce a strong T-cell response in the stomach, whereas the response within the duodenum has been poorly characterized. Furthermore, it remains to be elucidated whether the T-cell response may contribute to ulcer formation in the host. In this study, the frequency of different T-cell subsets, their degree of activation and expression of co-stimulatory receptors in biopsies from the duodenum as well as the antrum were studied by immunohistochemistry and flow cytometry. It was also evaluated whether there are differences in the T-cell responses between duodenal ulcer patients and asymptomatic carriers that might explain why only 10-15% of the infected subjects develop duodenal ulcers. The frequencies of CD4+, CD8+ and CD45RO+, i.e. memory T-cells, were significantly increased in the antrum, and the number of CD25+ cells was considerably higher in both the antrum and duodenum of duodenal ulcer patients and asymptomatic carriers as compared to uninfected individuals. Interestingly, the levels of immunosuppressive CTLA-4+ cells were significantly higher in the duodenum of duodenal ulcer patients, as compared to the asymptomatic carriers. H. pylori cause activation of T-cells in the duodenum as well as in the stomach. Our observation of higher levels of CTLA-4+ cells in the duodenum of duodenal ulcer patients than in the asymptomatic carriers suggests that a suppressive T-cell response may be related to the development of duodenal ulcers.

Endoscopic assessment and grading of Barrett's esophagus using magnification endoscopy and narrow-band imaging: accuracy and interobserver agreement of different classification systems (with videos)

BACKGROUND Three different classification systems for the evaluation of Barretts esophagus (BE) using magnification endoscopy (ME) and narrow-band imaging (NBI) have been proposed. Until now, no comparative and external evaluation of these systems in a clinical-like situation has been performed. OBJECTIVE To compare and validate these 3 classification systems. DESIGN Prospective validation study. SETTING Tertiary-care referral center. Nine endoscopists with different levels of expertise from Europe and Japan participated as assessors. PATIENTS Thirty-two patients with long-segment BE. INTERVENTIONS From a group of 209 standardized prospective recordings collected on BE by using ME combined with NBI, 84 high-quality videos were randomly selected for evaluation. Histologically, 28 were classified as gastric type mucosa, 29 as specialized intestinal metaplasia (SIM), and 27 as SIM with dysplasia/cancer. Assessors were blinded to underlying histology and scored each video according to the respective classification system. Before evaluation, an educational set concerning each classification system was carefully studied. At each assessment, the same 84 videos were displayed, but in different and random order. MAIN OUTCOME MEASUREMENTS Accuracy for detection of nondysplastic and dysplastic SIM. Interobserver agreement related to each classification. RESULTS The median time for video evaluation was 25 seconds (interquartile range 20-39 seconds) and was longer with the Amsterdam classification (P < .001). In 65% to 69% of the videos, assessors described certainty about the histology prediction. The global accuracy was 46% and 47% using the Nottingham and Kansas classifications, respectively, and 51% with the Amsterdam classification. The accuracy for nondysplastic SIM identification ranged between 57% (Kansas and Nottingham) and 63% (Amsterdam). Accuracy for dysplastic tissue was 75%, irrespective of the classification system and assessor expertise level. Interobserver agreement ranged from fair (Nottingham, κ = 0.34) to moderate (Amsterdam and Kansas, κ = 0.47 and 0.44, respectively). LIMITATION No per-patient analysis. CONCLUSIONS All of the available classification systems could be used in a clinical-like environment, but with inadequate interobserver agreement. All classification systems based on combined ME and NBI, revealed substantial limitations in predicting nondysplastic and dysplastic BE when assessed externally. This technique cannot, as yet, replace random biopsies for histopathological analysis.

Percutaneous implantation of gastric electrodes - a novel technique applied in animals and in patients.

Abstract  Temporary electrodes implanted under general anaesthesia, or via an oral or percutaneous endoscopic gastrostomy route have been used for testing of gastric electrical stimulation (GES). We have developed a principle for percutaneous electrode implantation. Leads were constructed so that the tip could be anchored to the gastric submucosa under gastroscopic control. Acute experiments were performed in anaesthetized pigs. Three patients referred for nausea and/or vomiting and non‐established indications for GES (chronic intestinal pseudo‐obstruction, functional dyspepsia without gastroparesis) were evaluated. Electrode function was tested by recording and stimulation techniques. In the pigs, a slow‐wave (SW) rhythm (3 min−1) was recorded with decrease in frequency at the end of the experiments. In the patients, implantation time from start of gastroscopy to end of electrode placement was 12–20 min. Electrode distance varied from 12 to 45 mm. Gastric electromyography showed a regular SW rhythm of about 3 min−1. Antral pressure waves had intervals being multiples of the SW‐to‐SW time. With temporary GES for 7–9 days, weekly frequency of the referral symptoms decreased >80% in two patients and 33% in one patient. Temporary percutaneous gastric leads can easily be implanted and may be used for testing of GES and study of gastric electrophysiology.

Esophageal barrier function and tight junction expression in healthy subjects and patients with gastroesophageal reflux disease: functionality of esophageal mucosa exposed to bile salt and trypsin in vitro.

Abstract Background and aims. Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function. However, the influence of acid and/or bile acids on human esophageal epithelial barrier function and the tight junction (TJ) proteins has not been fully elucidated. The aim of the study is to investigate the esophageal barrier function and TJ expression in healthy subjects and patients with GERD. The functionality of esophageal mucosa exposed to bile salt deoxycholic acid (DCA) and trypsin has been studied in vitro. Material and methods. Endoscopic biopsies from healthy controls and patients with GERD-related symptom with endoscopic erosive signs, as well as esophageal mucosa taken from patients undergoing esophagectomy were evaluated in Ussing chambers and by western blot and immunohistochemistry. Results. The esophageal epithelium from GERD patients had lower electrical resistance and higher epithelial currents than controls. Claudin-1 and -4 were significantly decreased in GERD patients. The bile salt DCA in the low concentration of 1.5 mM and trypsin increased the resistance and claudin-1 expression, while the higher concentration of 2.5 mM DCA and trypsin decreased the resistance and the claudin-3, -4 and E-cadherin expressions. Conclusion. In addition to acidic reflux, duodenal reflux components, such as bile salts and trypsin, have the potential to disrupt the esophageal barrier function, partly by modulating the TJ proteins. However, the expression of TJ is dependent on both the refluxed material as well as the concentration of the bile salt.

Angiotensin II receptors are expressed and functional in human esophageal mucosa

Only few studies have been devoted to the actions of the renin-angiotensin system (RAS) in the human gastrointestinal tract. The present study was undertaken to elucidate the expression and action of RAS in the human esophageal mucosa. Mucosal specimens with normal histological appearance were obtained from healthy subjects undergoing endoscopy and from patients undergoing esophagectomy due to neoplasm. Gene and protein expressions of angiotensin II (Ang II) receptor type 1 (AT(1)) and type 2 (AT(2)) and angiotensin-converting enzyme (ACE) were analyzed. In vivo functionality in healthy volunteers was reflected by assessing transmucosal potential difference (PD). Ussing chamber technique was used to analyze the different effects of Ang II on its AT(1) and AT(2) receptors. Immunoreactivity to AT(1) and AT(2) was localized to stratum superficiale and spinosum in the epithelium. ACE, AT(1), and AT(2) were found in blood vessel walls. Transmucosal PD in vivo increased following administration of the AT(1) receptor antagonist candesartan. In Ussing preparations mean basal transmural PD was -6.4 mV, epithelial current (I(ep)) 34 muA/cm(2), and epithelial resistance (R(ep)) 321 Omega.cm(2). Serosal exposure to Ang II increased PD as a result of increased I(ep), whereas R(ep) was constant. Ang II given together with the selective AT(1)-receptor antagonist losartan, or AT(2) agonist C21 given alone, resulted in a similar effect. Ang II given in presence of the AT(2)-receptor antagonist PD123319 did not influence PD, but I(ep) decreased and R(ep) increased. In conclusion, Ang II receptors and ACE are expressed in the human esophageal epithelium. The results suggest that AT(2)-receptor stimulation increases epithelial ion transport, whereas the AT(1) receptor inhibits ion transport and increases R(ep).

Angiotensin II induced contraction of rat and human small intestinal wall musculature in vitro

Background:  Angiotensin II (Ang II) is a well‐known activator of smooth muscle in the vasculature but has been little explored with regard to intestinal wall muscular activity. This study investigates pharmacological properties of Ang II and expression of its receptors in small‐intestinal smooth muscle from rats and humans.

Expression of protease-activated-receptor 2 (PAR-2) in human esophageal mucosa

Objective. The role of duodenal reflux in gastroesophageal reflux disease (GERD) containing bile salts and pancreatic enzymes (with special attention to trypsin) is still under discussion. Proteinase-activated receptors (PARs) are a novel family and PAR-2 is a unique member of this family because it is activated by trypsin. The aim of the present study was to examine the presence and the position of the PAR-2 receptor in human esophageal mucosa in different subgroups of GERD. Material and methods. Distal biopsies taken from healthy controls, patients with erosive reflux disease (ERD), patients with specialized intestinal metaplasia (SIM) and adenocarcinoma were analyzed for the PAR-2 receptor with reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Results. Gene transcripts for the PAR-2 receptor were found in all groups, with increased levels in SIM patients compared to controls. However, this visual pattern was not seen for the protein expression of the PAR-2 receptor showing no apparent quantitative differences between the groups. Immunohistochemistry revealed distinct staining for the PAR-2 receptor in the luminal part of the esophageal epithelium. Conclusions. The localization of the PAR-2 receptor indicates that the receptor can be cleaved and activated by trypsin in duodenogastric esophageal refluxate. The data thus suggest that the trypsin-PAR-2 pathway may be involved in the pathogenesis of GERD.

Angiotensin II receptor expression and relation to Helicobacter pylori-infection in the stomach of the Mongolian gerbil

BackgroundThe role of the renin-angiotensin system in gastric physiology and disease has as yet been sparsely explored. The first aim of the study was to investigate the baseline presence and location of angiotensin II receptors (AT1R and AT2R) in the stomach of the Mongolian gerbil. A second aim was to elucidate whether the presence of H. pylori infection is associated with changes in the expression of these receptors.MethodsH. pylori-negative and H. pylori-infected (strain SS1 or TN2GF4) male Mongolian gerbils were investigated. The stomachs were examined at six or 12 months after inoculation by the use of immunohistochemistry, western blot and microscopic morphometry.ResultsAT1R and AT2R were located in a variety of cells in the gerbil gastric wall, including a subpopulation of endocrine cells in the antral mucosa and inflammatory cells infiltrating H. pylori-infected stomachs. Gerbils infected with the SS1 strain showed a significantly increased antral AT1R protein expression and an increased number of infiltrating polymorphonuclear leucocytes (PMNs) at 12 months. The AT1R protein expression correlated with the number of PMNs and the antral expression of myeloperoxidase.ConclusionsAngiotensin II receptors are present in a variety of cells in the gastric wall of the Mongolian gerbil. The results indicate an influence dependent on the H. pylori strain on the gastric AT1R expression and a relationship between gastric AT1R expression and mucosal PMNs infiltration.