Esophageal functional impairments in experimental eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is an emerging chronic esophageal disease. Despite the increasing diagnosis of EoE globally, the causes of EoE and other esophageal eosinophilic disorders are not clearly understood. EoE pathology includes accumulation of inflammatory cells (e.g., eosinophils, mast cells), characteristic endoscopic features (e.g., furrows, the formation of fine concentric mucosal rings, exudates), and functional impairments (e.g., esophageal stricture, dysmotility). We hypothesized that the esophageal structural pathology and functional impairments of EoE develop as a consequence of the effector functions of the accumulated inflammatory cells. We analyzed eosinophils (anti-major basic protein immunostaining), esophageal stricture (X-ray barium swallowing), and esophageal motility (isometric force) in two established transgenic murine models of EoE (CD2-IL-5 and rtTA-CC10-IL-13) and a novel eosinophil-deficient model (ΔdblGATA/CD2-IL-5). Herein, we show the following: 1) CD2-IL-5 and doxycycline (DOX)-induced rtTA-CC10-IL-13 mice have chronic eosinophilic and mast cell esophageal inflammation; 2) eosinophilic esophageal inflammation promotes esophageal stricture in both transgenic murine models; 3) the eosinophil-deficient ΔdblGATA/CD-2-IL-5 mice were protected from the induction of stricture, whereas the eosinophil-competent CD2-IL-5 mice develop esophageal stricture; 4) esophageal stricture is not reversible in DOX-induced rtTA-CC10-IL-13 mice (8 wk DOX followed by 8 wk no-DOX); and 5) IL-5 transgene-induced (CD2-IL-5) EoE evidences esophageal dysmotility (relaxation and contraction) that is independent of the eosinophilic esophageal inflammation: CD2-IL-5 and ΔdblGATA/CD2-IL-5 mice have comparable esophageal dysmotility. Collectively, our present study directly implicates chronic eosinophilic inflammation in the development of the esophageal structural impairments of experimental EoE.     

Significance of para-esophageal lymph nodes in food or aeroallergen-induced iNKT cell-mediated experimental eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder driven by food hypersensitivity; however, the specific foods and mechanisms involved are unclear. In patients with EoE, we have found that hypersensitivities to corn and peanuts are the most common. Accordingly, we sensitized and exposed mice either intranasally or intragastrically with corn or peanut extract or saline. Esophageal eosinophilia, the genes of eosinophil-directed cytokines, and allergen-induced antibodies were examined in mice challenged with corn or peanut extract or saline. A high number of esophageal lamina propria eosinophils as well as eosinophilic microabscesses, intraepithelial eosinophils, extracellular eosinophilic granules, thickened and disrupted epithelial mucosa, and mast cell hyperplasia were observed in the esophagus of peanut or corn allergen-challenged mice. Mechanistic analysis indicated that para-esophageal lymph nodes might be critical in the trafficking of eosinophils to the esophagus and in EoE association to airway eosinophilia. Furthermore, experimentation with gene-targeted mice revealed that peanut allergen-induced EoE was dependent on eotaxin and invariant natural killer T (iNKT) cells, as CD1d and eotaxin-1/2 gene-deficient mice were protected from disease induction. Thus we provide evidence that para-esophageal lymph nodes are involved in food- or aeroallergen-induced eosinophilia and patchy EoE pathogenesis, likely a mechanism dependent on eotaxins and iNKT cells.     

A Pilot Study of Omalizumab in Eosinophilic Esophagitis

Eosinophilic disorders of the gastrointestinal tract are an emerging subset of immune pathologies within the spectrum of allergic inflammation. Eosinophilic Esophagitis (EoE), once considered a rare disease, is increasing in incidence, with a rate of over 1 in 10,000 in the US, for unknown reasons. The clinical management of EoE is challenging, thus there is an urgent need for understanding the etiology and pathophysiology of this eosinophilic disease to develop better therapeutic approaches. In this open label, single arm, unblinded study, we evaluated the effects of an anti-IgE treatment, omalizumab, on local inflammation in the esophagus and clinical correlates in patients with EoE. Omalizumab was administered for 12 weeks to 15 subjects with long standing EoE. There were no serious side effects from the treatment. Esophageal tissue inflammation was assessed both before and after therapy. After 3 months on omalizumab, although tissue Immunoglobulin E (IgE) levels were significantly reduced in all but two of the subjects, we found that full remission of EoE, which is defined as histologic and clinical improvement only in 33% of the patients. The decrease in tryptase-positive cells and eosinophils correlated significantly with the clinical outcome as measured by improvement in endoscopy and symptom scores, respectively. Omalizumab-induced remission of EoE was limited to subjects with low peripheral blood absolute eosinophil counts. These findings demonstrate that in a subset of EoE patients, IgE plays a role in the pathophysiology of the disease and that anti-IgE therapy with omalizumab may result in disease remission. Since this study is open label there is the potential for bias, hence the need for a larger double blind placebo controlled study. The data presented in this pilot study provides a foundation for proper patient selection to maximize clinical efficacy.

Trial Registration

ClinicalTrials.gov NCT01040598     

Esophageal Microbiome in Eosinophilic Esophagitis

Objective

The microbiome has been implicated in the pathogenesis of a number of allergic and inflammatory diseases. The mucosa affected by eosinophilic esophagitis (EoE) is composed of a stratified squamous epithelia and contains intraepithelial eosinophils. To date, no studies have identified the esophageal microbiome in patients with EoE or the impact of treatment on these organisms. The aim of this study was to identify the esophageal microbiome in EoE and determine whether treatments change this profile. We hypothesized that clinically relevant alterations in bacterial populations are present in different forms of esophagitis.

Design

In this prospective study, secretions from the esophageal mucosa were collected from children and adults with EoE, Gastroesophageal Reflux Disease (GERD) and normal mucosa using the Esophageal String Test (EST). Bacterial load was determined using quantitative PCR. Bacterial communities, determined by 16S rRNA gene amplification and 454 pyrosequencing, were compared between health and disease.

Results

Samples from a total of 70 children and adult subjects were examined. Bacterial load was increased in both EoE and GERD relative to normal subjects. In subjects with EoE, load was increased regardless of treatment status or degree of mucosal eosinophilia compared with normal. Haemophilus was significantly increased in untreated EoE subjects as compared with normal subjects. Streptococcus was decreased in GERD subjects on proton pump inhibition as compared with normal subjects.

Conclusions

Diseases associated with mucosal eosinophilia are characterized by a different microbiome from that found in the normal mucosa. Microbiota may contribute to esophageal inflammation in EoE and GERD.     

Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE)

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl₂ differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.     

Roles of microRNAs in inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract that mainly affects young people. IBD is associated with various gastrointestinal symptoms, and thus, affects the quality of life of patients. Currently, the pathogenesis of IBD is poorly understood. Although intestinal bacteria and host immune response are thought to be major factors in its pathogenesis, a sufficient explanation of their role in its pathophysiologic mechanism has not been presented. MicroRNAs (miRNAs), which are small RNA molecules that regulate gene expression, have gained attention as they are known to participate in the molecular interactions of IBD. Recent studies have confirmed the important role of miRNAs in targeting certain molecules in signaling pathways that regulate the homeostasis of the intestinal barrier, inflammatory reactions, and autophagy of the intestinal epithelium. Several studies have identified the specific miRNAs associated with IBD from colon tissues or serum samples of IBD patients and have attempted to use them as useful diagnostic biomarkers. Furthermore, some studies have attempted to treat IBD through intracolonic administration of specific miRNAs in the form of nanoparticle. This review summarizes the latest findings on the role of miRNAs in the pathogenesis, diagnosis, and treatment of IBD.     

Clinical findings,early endoscopy,and multivariate analysis in patients bleeding from the upper gastrointestinal tract.

A simple system has been developed to identify patients with upper gastrointestinal tract haemorrhage who run a high risk of continued bleeding or rebleeding. The system is based on six items of patient data available at or soon after arrival in hospital. It was evaluated in a prospective study of 66 patients with upper gastrointestinal tract haemorrhage. Over half of the patients classified by the system into a high-risk category either continued bleeding or rebled after apparent cessation (as against one out of 33 patients in the low-risk category). The high-rish group also had a higher mortality (21%) than those in the low-risk group (nil). The addition or subtraction of early endoscopic findings made little difference to the accuracy of prognosis.     

Molecular Profiling of Thin-Prep FNA Samples in Assisting Clinical Management of Non-Small-Cell Lung Cancer

The discovery of new target treatments for NSCLC has led to a search for new genetic and epigenetic markers able to selectively predict response to these new drugs. Somatic mutations in EGFR and KRAS genes are routinely analyzed to predict response to tyrosine kinase inhibitors (TKIs), used in the treatment of NSCLC patients, whose efficacy depend on the presence or the absence of specific mutations. MicroRNA (miRNA) expression evaluation has been recently analyzed because of the involvement of these molecules in lung cancer pathogenesis and in drug resistance. Only 30 % of NSCLC patients present a resectable stage at time of diagnosis so tissue samples cannot be the only starting material for genetic and epigenetic analysis. Therefore, the possibility to use cytological sampling already used for diagnosis also for molecular testing is emerging. The aim of this study was to evaluate for the first time in lung cancer the use of liquid-based cytology both for EGFR and KRAS mutational testing and for the expression trend of some miRNAs involved in lung cancer pathogenesis: miR-21, miR-155, miR-7, and let7a. We enrolled 20 fine-needle aspirate (FNA) samples diagnosed as NSCLC, 10 FNAs without neoplastic cells, and tissue samples coming from 5 of the 20 patients who underwent surgery after FNA NSCLC diagnosis. All Thin-Prep processed FNA samples were evaluable for DNA and RNA analysis and results were compared with those of the small group of patients whose matched tumor histology was available. The mutational status of the EGFR and KRAS genes and the expression profile of the selected miRNA showed comparable results between FNA samples and histological tissues. Our results underline that cytological samples could give the same genetic information as that obtained from histological specimens and so could be collected to create a nucleic acids bank.     

Diagnosis of deep‐seated lymphomas by endoscopic ultrasound‐guided fine needle aspiration combined with flow cytometry

A. Stacchini, P. Carucci, D. Pacchioni, G. Accinelli, A. Demurtas, S. Aliberti, M. Bosco, M. Bruno, A. Balbo Mussetto, M. Rizzetto, G. Bussolati and C. De Angelis
Diagnosis of deep‐seated lymphomas by endoscopic ultrasound‐guided fine needle aspiration combined with flow cytometry Objective: Although endoscopic ultrasound combined with fine needle aspiration (EUS‐FNA) is rapidly becoming the preferred diagnostic approach for the sampling and diagnosis of gastrointestinal and mediastinal malignancies, there are limited data as to its use in the diagnosis of lymphoproliferative disorders. Therefore, we carried out a retrospective evaluation of the performance of EUS‐guided FNA combined with flow cytometry (FC) as a tool to improve overall sensitivity and specificity in the diagnosis of lymphoma. Methods: Of 1560 patients having EUS‐guided FNA during the period of the study, a total of 56 patients were evaluated by cytology with FC after EUS‐FNA. There was adequate material to perform FC analysis for all but one case. Results: EUS‐FNA‐FC gave a diagnosis of lymphoma in 11 cases and of reactive lymphadenopathy in 20. A specific histological type was defined by FC alone in eight cases. The remaining cases were diagnosed later by cytology and cell block sections: 13 carcinomas, nine granulomatous lymphadenopathies and one mediastinal extramedullary haematopoiesis. One case was considered only suspicious for lymphoma on cytology and FC but was not confirmed on molecular analysis and one had insufficient material for FC. Conclusions: Our results show that a combination of EUS‐FNA‐FC is a feasible and highly accurate method, which may be used for the diagnosis and subtyping of deep‐seated lymphoma, providing a significant improvement to cytomorphology alone both for diagnosis and treatment planning, as long as immunocytochemistry is available for non‐lymphoma cases.     

Role of faecal gas analysis for the diagnosis of IBD

The diagnosis of IBD (inflammatory bowel disease) is based on the clinical evaluation of symptoms and signs leading to a series of investigations. The investigations used are often unpleasant for patients; they are invasive, costly and potentially dangerous. Patients often report that the odour of flatus, or the gas emitted from faeces, is abnormal during a flare of their IBD. Our group has characterized the VOCs (volatile organic compounds) in the headspace gas emitted from faecal samples from healthy subjects, from patients with infectious diarrhoea and from those with Crohn's disease or ulcerative colitis, both in relapse and remission. Painstaking analysis of gas chromatography-MS data (VOC profiling) has revealed patterns of compounds that are strongly associated with specific infectious diseases and with IBD. These compounds represent a change in the microflora and/or the metabolism of bacteria and/or the epithelium in disease states. These profiles offer a potential for rapid non-invasive assessment of a range of infectious and non-infectious gastrointestinal diseases. The study of VOCs may lead to a better understanding of the pathogenesis of IBD.     

Animal models of gastrointestinal inflammation and cancer

Inflammation and cancer are the two major disorders in the gastrointestinal tract. They are causally related in their pathogenesis. It is important to study animal models' causal relationship and, in particular, to discover new therapeutic agents for such diseases. There are several criteria for these models in order to make them useful in better understanding the etiology and treatment of the said diseases in humans. In this regard, animal models should be similar as possible to human diseases and also be easy to produce and reproducible and also economic to allow a continuous replication in different laboratories. In this review, we summarize the various animal models for inflammatory and cancerous disorders in the upper and lower gastrointestinal tract. Experimental approaches are as simple as by giving a single oral dose of alcohol or other noxious agents or by injections of multiple dosages of ulcer inducing agents or by parenteral administration or in drinking water of carcinogens or by modifying the genetic makeups of animals to produce relatively long-term pathological changes in particular organs. With these methods they could induce consistent inflammatory responses or tumorigenesis in the gastrointestinal mucosa. These animal models are widely used in laboratories in understanding the pathogenesis as well as the mechanisms of action for therapeutic agents in the treatment of gastrointestinal inflammation and cancer.     

Combined analysis of chromosomal instabilities and gene expression for colon cancer progression inference

Background

The human body plays host to a vast array of bacteria, found in oral cavities, skin, gastrointestinal tract and the vagina. Some bacteria are harmful while others are beneficial to the host. Despite the availability of many methods to identify bacteria, most of them are only applicable to specific and cultivable bacteria and are also tedious. Based on high throughput sequencing technology, this work derives 16S rRNA sequences of bacteria and analyzes probiotics and pathogens species.

Results

We constructed a database that recorded the species of probiotics and pathogens from literature, along with a modified Smith-Waterman algorithm for assigning the taxonomy of the sequenced 16S rRNA sequences. We also constructed a bacteria disease risk model for seven diseases based on 98 samples. Applicability of the proposed platform is demonstrated by collecting the microbiome in human gut of 13 samples.

Conclusions

The proposed platform provides a relatively easy means of identifying a certain amount of bacteria and their species (including uncultivable pathogens) for clinical microbiology applications. That is, detecting how probiotics and pathogens inhabit humans and how affect their health can significantly contribute to develop a diagnosis and treatment method.     

Cytodiagnosis of in situ and early carcinoma of the upper gastrointestinal tract

Three cases of adenocarcinoma of the stomach, two in situ and one superficially invasive, and one of superficially invasive squamous-cell carcinoma of the esophagus are presented to illustrate the problems encountered in the diagnosis of early lesions of the upper gastrointestinal (GI) tract and the contribution that cytodiagnosis can make. The symptomology and roentgenographic findings in these cases were largely nonspecific. While endoscopic biopsies were repeatedly negative in three of the four cases, endoscopic brushing cytology consistently indicated the presence of a malignancy. Surgery was finally performed on the basis of the cytologic findings, confirming the presence of early malignancy. The cytologic findings, with histologic correlations, are presented in an effort to define some specific criteria for the diagnosis of early malignancy of the upper GI tract.     

Effect of melittin on mice stomach

Melittin, the main bee venom component, has many positive biological effects and a relatively low toxicity in various cell types. However, there is no evidence of the effect of melittin on gastrointestinal cells. In the present study, we investigated the histological and immuonohistochemical effects of melittin on mice stomach. Adult male mice (Albino Swiss) were randomly divided into two groups (7 mice for each group): control group and melittin only treated group (10 and 40 μg/kg). These mice were sacrificed, then samples from the stomach were collected and prepared for histopathological studies by using alcian blue stain and immuonohistochemical studies by using smooth muscle actin (SMA) antibody. Treatment with melittin alone do not cause any harmful effect on the stomach tissue where the microscopic examination of Alcian blue stained section showed the normal distribution of the mucous secreting cells of the stomach tissues. On other hand, no changes were observed on smooth muscle cells. This study demonstrated the safety of using melittin on gastrointestinal tissues if used in definite dose and for suitable duration, which offers an opportunity for its use as a treatment for many diseases of the gastrointestinal tract.     

21例奶蛋白过敏患儿临床特征分析及诊治方法

目的：探讨奶蛋白过敏患儿的临床特点及诊治方法。方法：收集2012年2月-2013年2月我院接诊的21例奶蛋白过敏患儿的临床资料,将临床表现、特异性皮肤点刺试验、回避试验等结果进行总结分析。结果：患儿皮肤粘膜、消化道等症状出现时间较早,湿疹症状出现最多（71．43％）,仅有1例患儿出现发育迟缓（4．76％）;皮肤点刺试验奶蛋白阳性者12例（57．14％）,回避试验阳性率为100．00％;经过改善饮食及对症治疗,21例患儿皮肤粘膜、消化道、呼吸道系统等症状一般在4周内消失,发育迟缓在正规治疗2．4周开始缓解,8周内消失。结论：奶蛋白过敏缺乏特异症状表现,临床在出现症状后加强实验室检查。结合详细病史进行确诊,才能减少误诊的发生,及时治疗,保证患儿的正常生长发育。     

Probiotics--an alternative treatment for various diseases

Modulating the microbiota of the gastrointestinal tract through probiotics is an alternative to the conventional treatment of various diseases, based on synthetic drugs. The lifestyle, nutrition and stress of the present modern society could be among the factors responsible for modifications in the intestinal microbiota, correlated with specific diseases. The present study describes the positive effects of probiotics use, with special reference to the yeasts use in several frequently encountered diseases, such as hypercholesterolemia, the irritable bowel syndrome, gastritis and several uro-genital disorders.     

胃食管反流病与胃肠道微生态关系的研究进展

胃食管反流病(gastroesophageal reflux disease, GERD)是由多种原因引起的功能性胃肠疾病, 发病率逐年上升, 目前其发病机制尚不完全清楚。GERD的典型症状是胃灼热和反流。研究发现GERD的发生发展与胃肠道微生态有密切的关系。胃肠道中有着人体内最复杂的微生态系统, 其微生态平衡是维持人体健康的关键因素。而胃肠道微生态失衡导致食管抗反流结构和功能受损、食管清除作用降低、食管黏膜抵御能力下降, 最终通过精神心理疾病、肥胖和糖尿病等引发或加重GERD。本文综述了GERD与胃肠道微生态的直接关系, 探讨GERD的危险因素与肠道菌群的关系, 并对GERD的微生态治疗进行了阐述。

     

粪菌移植治疗肠易激综合征研究进展

粪菌移植(faecal microbiota transplantation,FMT)是将从健康供体获得的粪便悬液转移至患者消化道,从而恢复肠道正常微生物组成和功能的一种治疗方法。近年来,FMT治疗肠易激综合征(irritable bowel syndrome,IBS)的疗效和经济效益越来越受到研究者的重视。这种方法已经成为艰难梭菌感染(Clostridium difficile infection,CDI)的成熟替代疗法。此外,FMT还在炎症性肠病(inflammatory bowel disease,IBD)和胃肠疾病以外的疾病,如心血管疾病、自身免疫性疾病和代谢综合征等多种疾病的治疗中取得了显著进展。关于FMT仍有许多未解的问题,需要在这一领域进行更多研究。本文就IBS的发病机制、FMT的疗效和安全性进行综述。