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The effects of cyclophosphamide on 5-hydroxytryptamine (5-HT) synthesis in the intestinal tissue of rats were investigated. Rats received 120 mg/kg cyclophosphamide intraperitoneally as a single administration, and kaolin and food intake was measured by an automatic monitoring apparatus. Ileal tissues were collected at either 24 or 72 h after administration. Cyclophosphamide caused a significant increase in kaolin intake at the acute and the delayed phases and was associated with a decrease in food intake, and body weight. Cyclophosphamide had no significant effect on intestinal mucosal morphology, or inducible nitric oxide synthase and cyclooxygenase-2 expression in the intestine. Cyclophosphamide significantly increased tryptophan hydroxylase 1 (TPH1) mRNA expression, number of anti-TPH antibody-positive cells, and 5-HT content in the intestine. Cyclophosphamide also significantly increased the expression of Tac1 mRNA, encoding preprotachykinin-1, which is a preprotein of substance P, and the number of anti-substance P antibody-positive cells in the intestine. Cyclophosphamide significantly increased Lgr5, Bmi1, and Atoh1 mRNA levels, which are markers for the proliferation and differentiation of stem cells. This study demonstrated that cyclophosphamide induced pica in rats, and potentiated 5-HT synthesis associated with hyperplasia of substance P-containing enterochromaffin cells without causing severe intestinal injury., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Objectives: Enterochromaffin (EC) cells have been associated with functional gastrointestinal disorders such as IBS. Recently, we found that glial cell-derived neurotrophic factor (GDNF)-rearranged during transfection (RET) localized in EC cells in human colonic epithelia. Here, we examine the role of GDNF-RET in the pathophysiology of diarrhoea-predominant irritable bowel syndrome (IBS-D)., Materials and Methods: GDNF was assessed by ELISA and immunohistochemistry in biopsies from IBS-D patients and healthy controls. Stress was induced by using a wrap-restraint stress (WRS) procedure to serve as an acute stress-induced IBS model. The function of GDNF-RET axis to intestinal stem cell (ISC) homeostasis, and EC cell numbers were assessed in vivo and in vitro., Results: GDNF-RET was expressed in EC cells in human colon. GDNF was significantly increased in IBS-D patients. WRS mice showed increased GDNF-RET levels in colon. WRS induced visceral hypersensitivity by expanding of ISC and differentiation of EC cell via GDNF-RET. Furthermore, GDNF-treated mice recapitulated the phenotype of WRS mice. In vitro, GDNF treatment amplified Wnt signal and increased serotonin levels in colonic organoids in a dose-dependent manner., Conclusions: We identified GDNF-RET was presented in colonic epithelium of patients with IBS-D. GDNF-RET played important roles in regulating ISC and EC cell differentiation. Our findings, thus, provide RET inhibitor as new therapeutic targets for treatment of patients with IBS-D., (© 2020 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Small intestinal neuroendocrine tumors (SI-NET) are serotonin-secreting well-differentiated neuroendocrine tumors of putative enterochromaffin (EC) cell origin. Recent studies recognize a subset of EC cells that is label-retaining at the +4 position in the crypt and functions as a reserve intestinal stem cell. Importantly, this +4 reserve EC cell subset not only contributes to regeneration of the intestinal epithelium during injury and inflammation but also to basal crypt homeostasis at a constant rate. The latter function suggests that the +4 EC cell subset serves as an active reserve stem cell via a constant rate of dedifferentiation. Characterization of early tumor formation of SI-NET, observed as crypt-based EC cell clusters in many cases of familial SI-NETs, suggests that the +4 active reserve EC cell subset is the cell of origin. This newly discovered active reserve stem cell property of EC cells can account for unique biological mechanisms and processes associated with the genesis and development of SI-NETs. The recognition of this property of the +4 active reserve EC cell subset may provide novel opportunities to explore NETs in the gastrointestinal tract and other organs.

Proton pump inhibitors (PPIs) are recommended as a first-line treatment for gastroesophageal reflux disease (GERD) and other acid related disorders. In recent years, concerns have been raised about the increasing prevalence of patients on long-term PPI therapy and inappropriate PPI use. It is well known that short-term PPI therapy is generally well tolerated and safe; however, their extensive long-term use is a major global issue. One of these long-standing concerns is PPI-induced gastrin elevation secondary to hypoacidity. Hypergastrinemia is believed to play a role in rebound hyperacidity when PPIs are discontinued resulting in induced dyspeptic symptoms that might result in the reinstitution of therapy. Gastrin exerts tropic effects in the stomach, especially on enterochromaffin-like (ECL) cells, and concerns have also been raised regarding the potential progression to dysplasia or tumor formation following long-term therapy. It is well known that a substantial number of patients on long-term PPI therapy can discontinue PPIs without recurrence of symptoms in deprescribing trials. What is unknown is how sustainable deprescribing should be undertaken in practice and how effective it is in terms of reducing long-term outcomes like adverse drug events, morbidity and mortality. Moreover, there is no clear consensus on when and how deprescribing strategies should be attempted in practice. This review sought to summarize the harms and benefits of long-term PPI therapy with special focus on gastrin elevation and its relation to deprescribing studies and future interventions that may improve PPI use., Competing Interests: The authors declare no conflict of interest.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

The role of nitric oxide (NO) in the changes in enterochromaffin cells and ileal 5-hydroxytryptamine (5-HT) content induced by a single i.p. administration of methotrexate was investigated in rats. Methotrexate significantly increased inducible NO synthase (iNOS) mRNA and protein expressions in the intestinal tissue at 96 h. Methotrexate also significantly caused hyperplasia of the enterochromaffin cells at 96 h; this was associated with a significant increase in 5-HT content. The methotrexate-induced hyperplasia of enterochromaffin cells and increase in 5-HT content were, however, completely suppressed by daily treatment with dexamethasone, and with N G -nitro-l-arginine methyl ester (l-NAME); this was not observed when meloxicam was administered. Histological examination showed slight but not pronounced mucosal injury, at 96 h after methotrexate administration. The methotrexate-induced decrease in body weight did not fully recover to the control level up to 96 h; however, the methotrexate-induced decrease in food/water intake slightly returned to the control level up to 96 h. l-NAME had no significant effect on methotrexate-induced body weight loss and anorexia. To conclude, the present study suggests that NO derived from methotrexate-induced iNOS plays a critical role in the mechanism of hyperplasia of enterochromaffin cells containing 5-HT in the intestinal tissue of rats., (Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Serotonin (5-hydroxytryptamine [5-HT]) is a key enteric signaling molecule that mediates various physiological processes in the gut. Enterochromaffin (EC) cells in the mucosal layer of the gut are the main source of 5-HT in the body and are situated in close proximity to the gut microbiota. In this study, we identify a pivotal role of TLR2 in 5-HT production in the gut. Antibiotic treatment reduces EC cell numbers and 5-HT levels in naive C57BL/6 mice, which is associated with downregulation of TLR2 expression but not TLR1 or TLR4. TLR2-deficient ( Tlr2 -/- ) and Myd88 -/- mice express lower EC cell numbers and 5-HT levels, whereas treatment with TLR2/1 agonist upregulates 5-HT production in irradiated C57BL/6 mice, which are reconstituted with Tlr2 -/- bone marrow cells, and in germ-free mice. Human EC cell line (BON-1 cells) release higher 5-HT upon TLR2/1 agonist via NF-κB pathway. Tlr2 -/- mice and anti-TLR2 Ab-treated mice infected with enteric parasite, Trichuris muris , exhibited attenuated 5-HT production, compared with infected wild-type mice. Moreover, excretory-secretory products from T. muris induce higher 5-HT production in BON-1 cells via TLR2 in a dose-dependent manner, whereby the effect of excretory-secretory products is abrogated by TLR2 antagonist. These findings not only suggest an important role of TLR2 in mucosal 5-HT production in the gut by resident microbiota as well as by a nematode parasite but also provide, to our knowledge, novel information on the potential benefits of targeting TLR2 in various gut disorders that exhibit aberrant 5-HT signaling., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Early childhood is a critical period for development, and early life stress may increase the risk of gastrointestinal diseases including irritable bowel syndrome (IBS). In rodents, neonatal maternal separation (NMS) induces bowel dysfunctions that resemble IBS. However, the underlying mechanisms remain unclear. Here we show that NMS induces expansion of intestinal stem cells (ISCs) and their differentiation toward secretory lineages including enterochromaffin (EC) and Paneth cells, leading to EC hyperplasia, increased serotonin production, and visceral hyperalgesia. This is reversed by inhibition of nerve growth factor (NGF)-mediated tropomyosin receptor kinase A (TrkA) signalling, and treatment with NGF recapitulates the intestinal phenotype of NMS mice in vivo and in mouse intestinal organoids in vitro. Mechanistically, NGF transactivates Wnt/β-catenin signalling. NGF and serotonin are positively correlated in the sera of diarrhea-predominant IBS patients. Together, our findings provide mechanistic insights into early life stress-induced intestinal changes that may translate into treatments for gastrointestinal diseases.

Idiopathic chronic diarrhea (ICD) is a common ailment affecting captive rhesus macaques ( Macaca mulatta). ICD cases are characterized by diarrhea in the absence of commonly identified diarrheal pathogens and multiple recurrences even after supportive therapy. Histologically, the disease is characterized by lymphoplasmacytic colitis. We identified 35 rhesus macaques euthanized for ICD during a 7-month period and described demographic, clinical, histologic, and immunologic commonalities. We found a trend of historic Campylobacter spp. and trichomonad infections. Furthermore, rhesus macaques with ICD demonstrated loss of normal colonic adherent bacterium, identified in this study as Helicobacter macacae; increased abundance of Pentatrichomonas hominis; and increased frequency of colonic serotonin-positive enterochromaffin cells. Interestingly, colonic and ileal T-helper cells of animals with ICD manifested decreased capacity for expression of certain cytokines, in particular interleukin (IL)-4 and IL-13. These data further describe a common ailment and suggest new avenues to identify complex interactions involved in the etiology of recurring diarrhea in young rhesus macaques.

Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells. IMPORTANCE The nonenveloped human adenovirus 41 causes diarrhea, vomiting, dehydration, and low-grade fever mainly in children under 2 years of age. Even though acute gastroenteritis is well described, how human adenovirus 41 causes diarrhea is unknown. In our study, we analyzed the effect of human adenovirus 41 infection on human enterochromaffin cells and found it stimulates serotonin secretion in the cells, which is involved in regulation of intestinal secretion and gut motility and can also activate enteric glia cells, which are found in close proximity to enterochromaffin cells in vivo This disruption of gut barrier homeostasis as maintained by these cells following human adenovirus 41 infection might be a mechanism in enteric adenovirus pathogenesis in humans and could indicate a possible serotonin-dependent cross talk between human adenovirus 41, enterochromaffin cells, and enteric glia cells., (Copyright © 2018 American Society for Microbiology.)

Background: Autoimmune gastritis (AIG) is a gastric pathologic condition affecting the mucosa of the fundus and the body and eventually leading to hypo-achlorhydria., Aims: We report our clinical and pathological experience with AIG., Methods: Data from patients with a diagnosis of AIG seen in the period January 2002-December 2012 were retrieved. Only patients with complete sets of biopsies were analyzed., Results: Data from 138 patients were available for analysis. Pernicious anemia was present in 25% of patients, iron deficiency anemia was found in 29.7% of patients, hypothyroidism in 23% of patients, type 1 diabetes in 7.9% of patients, and vitiligo in 2.8% of patients. Parietal cell antibodies were positive in 65% of patients, and no patient had serology positive for celiac disease. All gastric biopsies showed glandular atrophy associated with enterochromaffin-like (ECL)-cells hyperplasia, features limited to the mucosa of the fundus and body, and focal glandular intestinal metaplasia. Helicobacter pylori was negative in all cases., Conclusions: AIG was strongly associated with anemia; atrophy, intestinal metaplasia and ECL hyperplasia in the gastric fundus and body are hallmarks of this condition.

This study aimed to investigate the acute and chronic effect of methotrexate on the intestinal substance P metabolism after a single administration to rats. Methotrexate caused a significant increase in the number of substance P-containing cells in the ileal mucosa both at 24 and 96 h. Most of enterochromaffin cells expressing l-tryptophan hydroxylase contained substance P. The expression of Tac1 mRNA was increased by methotrexate at 24 h, but not at 96 h. Thus, methotrexate causes acute hyperplasia of enterochromaffin cells in the intestinal mucosa of rats with a transient increase in the production of substance P., (Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Advanced age is associated with a reduction in clinical visceral pain perception. However, the underlying mechanisms remain largely unknown. Previous studies have suggested that an abnormal interplay between mast cells, enterochromaffin (EC) cells, and afferent nerves contribute to nociception in gastrointestinal disorders. The aim of this study was to investigate how aging affects afferent sensitivity and neuro-immune association in the human bowel., Methods: Mechanical and chemical sensitivity of human bowel afferents were examined by ex vivo afferent nerve recordings. Age-related changes in the density of mast cells, EC cells, sensory nerve terminals, and mast cell-nerve micro-anatomical association were investigated by histological and immune staining., Key Results: Human afferents could be broadly classified into subpopulations displaying mechanical and chemical sensitivity, adaptation, chemo-sensitization, and recruitment. Interestingly human bowel afferent nerve sensitivity was attenuated with age. The density of substance P-immunoreactive (SP-IR) nerve varicosities was also reduced with age. In contrast, the density of ileal and colonic mucosal mast cells was increased with age, as was ileal EC cell number. An increased proportion of mast cells was found in close apposition to SP-IR nerves., Conclusions & Inferences: Afferent sensitivity in human bowel was reduced with advancing age. Augmentation of mast cells and EC cell numbers and the mast cell-nerve association suggest a compensatory mechanism for sensory neurodegeneration., (© 2016 The Authors. Neurogastroenterology & Motility Published by John Wiley & Sons Ltd.)

Aim: To study histidine decarboxylase (HDC) expression in normal and neoplastic gastric neuroendocrine cells in relationship to the main histamine metabolite., Methods: Control tissues from fundus (n = 3) and corpus (n = 3) mucosa of six patients undergoing operations for gastric adenocarcinoma, biopsy and/or gastric surgical specimens from 64 patients with primary gastric neuroendocrine tumours (GNETs), as well as metastases from 22 of these patients, were investigated using conventional immunohistochemistry and double immunofluorescence with commercial antibodies vs vesicular monoamine transporter 2 (VMAT-2), HDC and ghrelin. The urinary excretion of the main histamine metabolite methylimidazoleacetic acid (U-MeImAA) was determined using high-performance liquid chromatography in 27 of the 64 patients., Results: In the gastric mucosa of the control tissues, co-localization studies identified neuroendocrine cells that showed immunoreactivity only to VMAT-2 and others with reactivity only to HDC. A third cell population co-expressed both antigens. There was no co-expression of HDC and ghrelin. Similar results were obtained in the foci of neuroendocrine cell hyperplasia associated with chronic atrophic gastritis type A and also in the tumours. The relative incidence of the three aforementioned markers varied in the tumours that were examined using conventional immunohistochemistry. All of these GNETs revealed both VMAT-2 and HDC immunoreactivity, and their metastases showed an immunohistochemical pattern and frequency similar to that of their primary tumours. In four patients, increased U-MeImAA excretion was detected, but only two of the patients exhibited related endocrine symptoms., Conclusion: Human enterochromaffin-like cells appear to partially co-express VMAT-2 and HDC. Co-expression of VMAT-2 and HDC might be required for increased histamine production in patients with GNETs.

Objective: To investigate whether traditional Chinese herbal formula Yupingfeng (YPF) powder has an anti-inflammatory effect on colonic inflammation, and to explore the mechanism involved., Materials and Methods: YPF powder was orally administrated to trinitrobenzene sulfonic acid (TNBS)-induced colitis mice at the dose of 3, 6, and 12 g/kg/d for 7 consecutive days. Body weight, stool consistency, histopathological score, and myeloperoxidase (MPO) activity were tested to evaluate the effect of YPF powder on colonic inflammation while colonic enterochromaffin (EC) cell density and serotonin 5-hydroxytryptamine (5-HT) content were investigated to identify the effect of YPF powder on colonic 5-HT availability., Results: The results showed that the body weight of colitis mice was markedly decreased by 10, 12, 14, and 17% at 1, 3, 5, and 7 days (P < 0.05), whereas stool consistency score (3.6 vs. 0.4, P < 0.05), histopathological score (3.6 vs. 0.3, P < 0.05), and MPO activity (2.7 vs. 0.1, P < 0.05) in colitis mice were significantly increased compared to that of the normal mice; YPF powder treatment dose-dependently increased the body weight (7-13% increase) and decreased the stool consistency score (0.4-1.4 decrease), histopathological score (0.2-0.7 decrease), and MPO activity (0.1-0.9 decrease) in colitis mice. Colonic EC cell density (70% increase) and 5-HT content (40% increase) were markedly increased in colitis mice (P < 0.05), YPF powder treatment dose-dependently reduced EC cell density (20-50% decrease), and 5-HT content (5-27% decrease) in colitis mice., Conclusion: The findings demonstrate that the anti-inflammatory effect of YPF powder on TNBS - induced colitis may be mediated via reducing EC cell hyperplasia and 5-HT content. The important role of YPF powder in regulating colonic EC cell number and 5-HT content may provide an alternative therapy for colonic inflammation.

Neuroendocrine neoplasms (NEN) of the distal jejunum and ileum derive from serotonin-producing enterochromaffin (EC) cells. Due to their low proliferation rate and their infiltrative growth, they are often discovered at an advanced disease stage when metastasis has already occurred. The biology of these tumours is different from other NEN of the digestive tract. In order to standardise and improve diagnosis and therapy, the guidelines for the diagnosis and clinical management of jejuno-ileal NEN as well as for the management of patients with liver and other distant metastases from NEN were revised by the European Neuroendocrine Tumour Society (ENETS) in 2012. This review focuses on aspects relevant for surgical pathology.

Background & Aims: The alimentary tract contains a diffuse endocrine system comprising enteroendocrine cells that secrete peptides or biogenic amines to regulate digestion, insulin secretion, food intake, and energy homeostasis. Lineage analysis in the stomach revealed that a significant fraction of endocrine cells in the gastric corpus did not arise from Neurogenin3 (Neurog3)-expressing cells, unlike enteroendocrine cells elsewhere in the digestive tract. We aimed to isolate enriched serotonin-secreting and enterochromaffin-like (ECL) cells from the stomach and to clarify their cellular origin., Methods: We used Neurogenic differentiation 1 (NeuroD1) and Neurog3 lineage analysis and examined the differentiation of serotonin-producing and ECL cells in stomach tissues of NeuroD1-cre;ROSA(tdTom), tryptophan hydroxylase 1 (Tph1)-cyan fluorescent protein (CFP), c-Kit(wsh/wsh), and Neurog3Cre;ROSA(tdTom) mice by immunohistochemistry. We used fluorescence-activated cell sorting to isolate each cell type for gene expression analysis. We also performed RNA sequencing analysis of ECL cells., Results: Neither serotonin-secreting nor ECL cells of the corpus arose from cells expressing NeuroD1. Serotonin-secreting cells expressed a number of mast cell genes but not genes associated with endocrine differentiation; they did not develop in c-Kit(wsh/wsh) mice and were labeled with transplanted bone marrow cells. RNA sequencing analysis of ECL cells revealed high expression levels of many genes common to endocrine cells, including transcription factors, hormones, ion channels, and solute transporters but not markers of bone marrow cells., Conclusions: Serotonin-expressing cells of the gastric corpus of mice appear to be bone marrow-derived mucosal mast cells. Gene expression analysis of ECL cells indicated that they are endocrine cells of epithelial origin that do not express the same transcription factors as their intestinal enteroendocrine cell counterparts., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Olfactory receptors (ORs) are expressed in the olfactory epithelium, where they detect odorants, but also in other tissues with additional functions. Some ORs are even overexpressed in tumor cells. In this study, we identified ORs expressed in enterochromaffin tumor cells by RT-PCR, showing that single cells can co-express several ORs. Some of the receptors identified were already reported in other tumors, but they are orphan (without known ligand), as it is the case for most of the hundreds of human ORs. Thus, genes coding for human ORs with known ligands were transfected into these cells, expressing functional heterologous ORs. The in vitro stimulation of these cells by the corresponding OR odorant agonists promoted cell invasion of collagen gels. Using LNCaP prostate cancer cells, the stimulation of the PSGR (Prostate Specific G protein-coupled Receptor), an endogenously overexpressed OR, by β-ionone, its odorant agonist, resulted in the same phenotypic change. We also showed the involvement of a PI3 kinase γ dependent signaling pathway in this promotion of tumor cell invasiveness triggered by OR stimulation. Finally, after subcutaneous inoculation of LNCaP cells into NSG immunodeficient mice, the in vivo stimulation of these cells by the PSGR agonist β-ionone significantly enhanced metastasis emergence and spreading.

The adenomatous polyposis coli gene is a key tumor suppressor gene. Alterations in this gene have been found in most sporadic colon cancers; associated with familial adenomatous polyposis; and found in neoplasms of other organs, such as the liver, stomach, lung, breast, and cerebellar medulloblastoma. In the heterogeneous group of neuroendocrine neoplasms of the gastrointestinal tract, the involvement of adenomatous polyposis coli is debated, and only occasional reports found adenomatous polyposis coli alterations in foregut and midgut neuroendocrine neoplasms, with adenomatous polyposis coli mutations only in the latter. To elucidate the penetrance of adenomatous polyposis coli alterations in ileal neuroendocrine neoplasms, we performed DNA fragment analysis (loss of heterozygosity for 5q22-23 and 5q23) and sequencing on the mutation cluster region of the adenomatous polyposis coli gene on 30 ileal enterochromaffin cell neuroendocrine neoplasms. Adenomatous polyposis coli gene mutations were detected in 23% of cases (7/30); in particular, 57% were missense and 14%, nonsense/frameshift, all novel and different from those reported in colorectal or other cancers. Loss of heterozygosity analysis demonstrated a deletion frequency of 15% (4/27). No association was found with features of tumor progression. Our observations support the involvement of somatic adenomatous polyposis coli alterations in tumorigenesis of ileal enterochromaffin cell neuroendocrine neoplasms; the mechanisms of adenomatous polyposis coli gene inactivation appear to be different from those reported in other tumor types., (© 2013.)

History A 55-year-old man presented with chronic epigastric pain lasting for about 1 year and without fever or vomiting. The abdomen was soft and tender at physical examination. Laboratory tests revealed unremarkable liver function, normal hemoglobin level, and normal amylase level. White blood cell count was normal, and there was no inflammatory syndrome. The patient's medical history included pancreatic gastrinoma resected by means of left pancreatectomy 31 years before, hyperparathyroidism treated with subtotal parathyroidectomy 24 years before, and a slowly growing lung mass known for 9 years. Esophagogastroduodenoscopy was performed because of a suspected gastroduodenal ulcer. The results showed numerous small (<10 mm) gastric and duodenal ulcers and multiple 10-15-mm polypoid gastric masses. Contrast material-enhanced dual-phase multidetector row computed tomography (CT) of the chest and abdomen was performed with a 64-section CT scanner (LightSpeed VCT; GE Healthcare, Milwaukee, Wis). Technical parameters for CT were as follows: pitch, 0.98; section thickness and reconstruction interval, 1.25 mm; 120 kVp; and variable milliamperage determined by x-, y-, and z-axis dose modulation. After an unenhanced abdominal scan, iobitridol, a nonionic iodinated contrast agent containing 350 mg of iodine per milliliter (Xenetix 350; Guerbet, Aulnay-sousbois, France), was administered intravenously through a 16-18-gauge catheter. A 120-mL dose of the contrast agent was injected via an antecubital vein at a rate of 4 mL/sec. No oral contrast medium was administered. After preliminary unenhanced abdominal scanning, arterial and portal venous phase acquisitions were obtained 45 and 80 seconds after initiation of contrast medium injection.

The role of putative preneoplastic enterochromaffin-like cell lesions, either hyperplastic or dysplastic, in the genesis of type 1 enterochromaffin-like cell neuroendocrine tumors associated with type A chronic atrophic gastritis, their actual neoplastic risk, and their precise histogenetic mechanism deserve further clarification by specific histopathologic studies coupled with patient follow-up. A total of 100 patients with severe type A chronic atrophic gastritis, enterochromaffin-like cell hyperplasia, and antral G-cell hyperplasia were endoscopically and histologically followed up for a median of 90.1 months (total of 9118 person-months). Preneoplastic enterochromaffin-like cell lesions and newly developed neuroendocrine tumors were investigated histologically and histochemically, in parallel with enterochromaffin-like cell lesions found in nontumor mucosa of another 32 well-characterized and previously reported type 1 neuroendocrine tumors. Both neuroendocrine and nonneuroendocrine mucosa changes were analyzed and statistically evaluated. During follow-up, 7 of 100 patients developed neuroendocrine tumors: 5 were in a group of 20 cases with previous enterochromaffin-like cell dysplasia and 2 were among 80 cases showing only enterochromaffin-like cell hyperplasia throughout the study (hazard ratio, 20.7; P < .001). The severity of enterochromaffin-like cell hyperplasia at first biopsy, with special reference to linear hyperplasia with 6 chains or more per linear millimeter, also increased the risk of neuroendocrine tumor development during follow-up (hazard ratio, 13.0; P < .001). Enterochromaffin-like cell microinvasive dysplastic lesions arising at the epithelial renewal zone level, in connection with immature proliferating mucous-neck cells, were found to be linked to early intramucosal neuroendocrine tumor histogenesis. Both enterochromaffin-like cell dysplasia and severe hyperplasia indicate increased risk of neuroendocrine tumor development in type A chronic atrophic gastritis with hypergastrinemia/G-cell hyperplasia., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: Peutz-Jeghers (PJ) syndrome is a rare familial disorder with the autosomal transmission characterized by multiple intestinal polyps, mucocutaneous pigmentation and increased incidence of various malignancies. Some clinical manifestations of PJ syndrome may be associated with the serotonin secretion from the enterochromaffin cells (EC)., Objective: Since no data have been reported so far regarding EC cells in PJ polyps, the aim of our study was to quantitatively investigate EC population in hamartomatous intestinal polyps in patients with the PJ syndrome., Methods: The samples of surgically removed PJ polyps from family members with the PJ syndrome were collected during 34-year follow-up period. Formalin-fixed paraffin-embedded specimens of twenty-one PJ polyps were stained with HE, AB-PAS, Van Gieson, Fontana-Masson, FIF and Grimelius. For immunohistochemical analysis, the following antibodies were used: chromogranin A, serotonin, Ki-67, desmin, vimentin and cytokeratin in order to eliminate differential diagnostic possibilities and to confirm diagnosis of PJ polyps., Results: Strong EC cell hyperplasia was observed within the tissue of the investigated polyps. Statistical analysis demonstrated significantly higher content of EC cells in PJ polyps than in the normal ileal mucosa., Conclusion: Marked hyperplasia of EC cells within the PJ polyps may be the most important contributor to functional disorders in patients with the PJ syndrome.

Aim: To study an assessment of the number of enterochromaffin cells and expression of hydroxyindole-O-methyltransferase in colonic mucosa and urine excretion of 6-sulfatoxymelatonin in patients with ulcerative colitis., Methods: The study included 30 healthy subjects (group I-C), 30 patients with ulcerative proctitis [group II-ulcerative proctitis (UP)] and 30 patients with ulcerative colitis [group III-ulcerative colitis (UC)] in acute phases of these diseases. The number of enterochromaffin cells (EC) was estimated in rectal and colonic mucosa. Bioptates were assembled from many different parts of the large intestine. Immunorective cells collected from various parts of the colon were counted according to the Eurovision DAKO (Dako A/S, Copenhagen, Denmark) System in the range of 10 fields in each bioptate at × 200 magnification. The level of mRNA expression of hydroxyindole-O-methyltransferase (HIOMT) in colonic mucosa was estimated with RT-PCR. Urine 6-sulfatoxymelatonin (6-HMS) excretion was determined immunoenzymatically using an IBL (IBL International GmbH, Hamburg, Germany) kit (RE 54031)., Results: The number of EC cells in healthy subjects (C) was 132.40 ± 31.26. In patients of group II (UP) and group III (UC) the number of these cells was higher--225.40 ± 37.35 (P < 0.001) and--225.24 ± 40.50 (P < 0.001) respectively. Similar differences were related to HIOMT expression, which was 1.04 ± 0.36 in group C, 1.56 ± 0.56 (P < 0.01) in group UP and 2.00 ± 0.35 (P < 0.001) in group UC. Twenty-four hour 6-HMS urinary excretion was as follows: C--6.32 ± 4.95 μg/24 h, UP - 26.30 ± 7.29 μg/24 h (P < 0.01), UC--2.30 ± 12.56 μg/24 h (P < 0.001). A correlation between number of EC cells and 6-HMS excretion was noted in all groups: r = 0.766 in patients with UP, r = 0.703 with UC and r = 0.8551 in the control group; the correlation between the results is statistically significant., Conclusion: In the acute phases of both UP and UC, proliferation of EC cells and high expression of HIOMT and urine excretion of 6-HMS is noted. These changes may represent a beneficial response in the anti-inflammatory and defense mechanism.

Objective: We recently demonstrated that hypoxia, a key feature of IBD, increases enterochromaffin (EC) cell 5-HT secretion, which is also physiologically regulated by the ADORA2B mechanoreceptor. Since hypoxia is associated with increased extracellular adenosine, we wanted to examine whether this nucleotide amplifies HIF-1α-mediated 5-HT secretion., Design: The effects of hypoxia were studied on IBD mucosa, isolated IBD-EC cells, isolated normal EC cells and the EC cell tumor derived cell line KRJ-1. Hypoxia (0.5% O2) was compared to NECA (adenosine agonist), MRS1754 (ADORA2B receptor antagonist) and SCH442146 (ADORA2A antagonist) on HIF signaling and 5-HT secretion. Antisense approaches were used to mechanistically evaluate EC cells in vitro. PCR and western blot were used to analyze transcript and protein levels of HIF-1α signaling and neuroendocrine cell function. An animal model of colitis was evaluated to confirm hypoxia:adenosine signaling in vivo., Results: HIF-1α is upregulated in IBD mucosa and IBD-EC cells, the majority (~90%) of which express an activated phenotype in situ. Hypoxia stimulated 5-HT release maximally at 30 mins, an effect amplified by NECA and selectively inhibited by MRS1754, through phosphorylation of TPH-1 and activation of VMAT-1. Transient transfection with Renilla luciferase under hypoxia transcriptional response element (HRE) control identified that ADORA2B activated HIF-1α signaling under hypoxic conditions. Additional signaling pathways associated with hypoxia:adenosine included MAP kinase and CREB. Antisense approaches mechanistically confirmed that ADORA2B signaling was linked to these pathways and 5-HT release under hypoxic conditions. Hypoxia:adenosine activation which could be reversed by 5'-ASA treatment was confirmed in a TNBS-model., Conclusion: Hypoxia induced 5-HT synthesis and secretion is amplified by ADORA2B signaling via MAPK/CREB and TPH-1 activation. Targeting ADORA2s may decrease EC cell 5-HT production and secretion in IBD.

Enterochromaffin (EC) cells in the gastrointestinal (GI) mucosa are the main source of serotonin (5-hydroxytryptamine (5-HT)) in the body. 5-HT is implicated in the pathophysiology of many GI disorders including functional and inflammatory bowel disorders. Herein we studied the role of interleukin 13 (IL-13) in EC cell biology by utilizing IL-13-deficient (IL-13-/-) mice and BON cells (a model for human EC cells). The numbers of EC cells and 5-HT amount were significantly lower in enteric parasite, Trichuris muris-infected IL-13-/- mice compared with the wild-type mice. This was accompanied with increased parasite burden in IL-13-/- mice. Treatment of naive and infected IL-13-/- mice with IL-13 increased EC cell numbers and 5-HT amount. BON cells expressed IL-13 receptor and in response to IL-13 produced more 5-HT. These results provide novel information on IL-13-mediated immunological control of 5-HT in the gut, which may ultimately lead to improved therapeutic opportunities in various GI disorders.

Introduction: Neurotransmitter imbalance is hypothesised as a pathogenetic mechanism in several bowel conditions. We previously reported increased 5-HT in the sigmoid mucosa of colon resected for complicated diverticular disease (DD). We aimed to identify if abnormal 5-HT expression is associated with symptoms of uncomplicated DD., Methods: This was a prospective, comparative study and follow-up survey of symptoms. We examined the differences in 5-HT between DD patients and controls, as well as the presence of bowel symptoms at time of endoscopy and also 2 years later. Sigmoid biopsies were collected at colonoscopy. Immunohistochemical staining for 5-HT cells was performed., Results: Eighty-seven patients were recruited, 37 (42.5 %) DD and 50 (57.5 %) controls. No patients underwent surgery. There was no significant difference in total mean number of 5-HT-positive cells in DD compared to controls or between patients and controls with abdominal symptoms. Forty-one patients (47.1 %) responded to questionnaires at median 57.8 months from biopsy. Eighteen (43.9 %) were DD and 23(56.1 %) controls. 5-HT counts showed no significant association to symptom persistence., Discussion: Although 5-HT expression has previously been found to be increased in complicated DD in whole bowel-resected specimens, the same is not confirmed on colonic mucosal biopsies. This raises the suggestion that 5-HT may be involved in the development of acute complications but may not be involved in the pathogenesis of chronic symptoms.

Glucose in the gut lumen activates gut endocrine cells to release 5-HT, glucagon-like peptide 1/2 (GLP-1/2), and glucose-dependent insulinotropic polypeptide (GIP), which act to change gastrointestinal function and regulate postprandial plasma glucose. There is evidence that both release and action of incretin hormones is reduced in type 2 diabetes (T2D). We measured cellular activation of enteroendocrine and enterochromaffin cells, enteric neurons, and vagal afferent neurons in response to intestinal glucose in a model of type 2 diabetes mellitus, the UCD-T2DM rat. Prediabetic (PD), recent-diabetic (RD, 2 wk postonset), and 3-mo diabetic (3MD) fasted UCD-T2DM rats were given an orogastric gavage of vehicle (water, 0.5 ml /100 g body wt) or glucose (330 μmol/100 g body wt); after 6 min tissue was removed and cellular activation was determined by immunohistochemistry for phosphorylated calcium calmodulin-dependent kinase II (pCaMKII). In PD rats, pCaMKII immunoreactivity was increased in duodenal 5-HT (P < 0.001), K (P < 0.01) and L (P < 0.01) cells in response to glucose; glucose-induced activation of all three cell types was significantly reduced in RD and 3MD compared with PD rats. Immunoreactivity for GLP-1, but not GIP, was significantly reduced in RD and 3MD compared with PD rats (P < 0.01). Administration of glucose significantly increased pCaMKII in enteric and vagal afferent neurons in PD rats; glucose-induced pCaMKII immunoreactivity was attenuated in enteric and vagal afferent neurons (P < 0.01, P < 0.001, respectively) in RD and 3MD. These data suggest that glucose sensing in enteroendocrine and enterochromaffin cells and activation of neural pathways is markedly impaired in UCD-T2DM rats.

Gastric carcinoid tumors comprise 7% of all gastrointestinal carcinoids and have significantly increased in incidence over the past few decades. Seventy to 80% of gastric carcinoids are type I, which usually are clinically asymptomatic and found incidentally at endoscopic evaluation for abdominal pain or anemia. In this review, advances in understanding the pathophysiology of type I gastric carcinoid are highlighted. In addition, various current diagnostic and treatment options are discussed. Although type I carcinoids generally hold a benign course, rigorous investigation is needed to ensure accurate diagnosis and optimal treatment. This includes appropriate diagnostic procedures and imaging and accurate staging of tumor. Tumor size, depth of invasion, presence of metastasis, and the tumor's gastrin dependency dictate treatment options. Appropriate treatments can consist of endoscopic resection, antrectomy, medical management, or frequent follow-up. This article provides a systematic method of evaluating and treating type I gastric carcinoid.

Objectives: Serotonin-producing tumors of the pancreas are rare endocrine neoplasms composed of enterochromaffin (EC) cells that have been mainly described in the literature as case reports. This study analyzes the clinicopathologic features of a series of pancreatic EC cell neoplasms and their similarities to and differences from intestinal EC cell tumors., Methods: The morphological, immunohistochemical, ultrastructural, and fluorescent in situ hybridization features of 15 pancreatic and 20 intestinal serotonin-producing neoplasms were compared. In addition, we reviewed the literature on pancreatic serotonin-producing tumors to better understand the clinicopathologic features of this rare tumor type., Results: The lack of substance P and acidic fibroblast growth factor immunoreactivity; the low immunohistochemical expression of CDX2, vesicular monoamine transporter 1, connective tissue growth factor, and prostatic acid phosphatase; the lack of S100-positive sustentacular cells; the strong expression of vesicular monoamine transporter 2; and peculiar ultrastructural features characterize pancreatic EC cell tumors and differentiate them from intestinal ones, although both categories show similar chromosome 18 cytogenetic alterations. The review of the literature indicates that pancreatic functioning tumors associated with the carcinoid syndrome arise in younger patients and are larger, more frequently malignant, and more aggressive neoplasms than pancreatic nonfunctioning ones., Conclusions: Pancreatic EC cell tumors show several different morphological features compared with related intestinal tumors despite similar cytogenetic alterations on chromosome 18.

Rotavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human EC tumor cells ex vivo and in vitro and are localized to both EC cells and infected enterocytes in the close vicinity of EC cells in the jejunum of infected mice. Purified NSP4, but not purified virus particles, evoked release of 5-HT within 60 minutes and increased the intracellular Ca²⁺ concentration in a human midgut carcinoid EC cell line (GOT1) and ex vivo in human primary carcinoid EC cells concomitant with the release of 5-HT. Furthermore, NSP4 stimulated a modest production of inositol 1,4,5-triphosphate (IP₃), but not of cAMP. RV infection in mice induced Fos expression in the NTS, as seen in animals which vomit after administration of chemotherapeutic drugs. The demonstration that RV can stimulate EC cells leads us to propose that RV disease includes participation of 5-HT, EC cells, the enteric nervous system and activation of vagal afferent nerves to brain structures associated with nausea and vomiting. This hypothesis is supported by treating vomiting in children with acute gastroenteritis with 5-HT₃ receptor antagonists.

Objectives: Serotonin (5-hydroxytryptamine, 5-HT) metabolism may be altered in gut disorders, including in the irritable bowel syndrome (IBS). We assessed in patients with IBS vs. healthy controls (HCs) the number of colonic 5-HT-positive cells; the amount of mucosal 5-HT release; their correlation with mast cell counts and mediator release, as well as IBS symptoms; and the effects of mucosal 5-HT on electrophysiological responses in vitro., Methods: We enrolled 25 Rome II IBS patients and 12 HCs. IBS symptom severity and frequency were graded 0-4. 5-HT-positive enterochromaffin cells and tryptase-positive mast cells were assessed with quantitative immunohistochemistry on colonic biopsies. Mucosal 5-HT and mast cell mediators were assessed by high-performance liquid chromatography or immunoenzymatic assay, respectively. The impact of mucosal 5-HT on electrophysiological activity of rat mesenteric afferent nerves was evaluated in vitro., Results: Compared with HCs, patients with IBS showed a significant increase in 5-HT-positive cell counts (0.37 ± 0.16% vs. 0.56 ± 0.26%; P=0.039), which was significantly greater in patients with diarrhea-predominant IBS vs. constipation-predominant IBS (P=0.035). Compared with HCs, 5-HT release in patients with IBS was 10-fold significantly increased (P < 0.001), irrespective of bowel habit, and was correlated with mast cell counts. A significant correlation was found between the mucosal 5-HT release and the severity of abdominal pain (r(s)=0.582, P=0.047). The area under the curve, but not peak sensory afferent discharge evoked by IBS samples in rat jejunum, was significantly inhibited by the 5-HT₃ receptor antagonist granisetron (P<0.005)., Conclusions: In patients with IBS, 5-HT spontaneous release was significantly increased irrespective of bowel habit and correlated with mast cell counts and the severity of abdominal pain. Our results suggest that increased 5-HT release contributes to development of abdominal pain in IBS, probably through mucosal immune activation.

Neuroendocrine tumors (NETs) of the stomach are the most frequent among all neuroendocrine neoplasms in the digestive tract. The diagnosis and classification are complicated by the fact that these tumors have to be categorized not only by common staging and grading but also according to their pathophysiological background (types). The types differ in their biological behaviour (aggressiveness) which influences therapeutic concepts. This article explains and summarizes the etiology and classification of gastric NETs and offers a precise concept for diagnosis and treatment to improve clinical outcome.

Background: Some patients with Zollinger-Ellison syndrome post curative gastrinoma resection continue to show gastric acid hypersecretion; however, the mechanism is unknown., Aim: The aim of this study was to prospectively study acid secretion following curative gastrinoma resection and analyze factors contributing in patients with Zollinger-Ellison syndrome., Methods: Fifty patients cured post gastrinoma resection were studied with serial assessments of acid secretory status, cure status and ECL-cell status/activity (with serial biopsies, CgA, urinary N-MIAA). Correlative analysis was performed to determine predictive factors., Results: Hypersecretion occurred in 31 patients (62%) and 14 had extreme-hypersecretion. There was an initial decline (3-6 months) in BAO/MAO, which then remained stable for eight years. Preoperative BAO correlated with the postoperative secretion, but not other clinical, tumoral, laboratory variables, the degree of postoperative acid suppression or type of antisecretory drug needed. Hypersecretors had greater postoperative ECL changes (P=0.005), serum CGA (P=0.009) and 24-h urinary N-MIAA (P=0.0038)., Conclusions: Post curative resection, gastric hypersecretion persists long term (mean 8 years) in 62% of patients and in 28% it is extreme, despite normogastrinemia. No preoperative variable except BAO correlates with postresection hypersecretion. The persistent increased ECL-cell extent post curative resection suggests prolonged hypergastrinemia can lead to changes in ECL-cells that are either irreversible in humans or sustained by unknown mechanisms not involving fasting hypergastrinemia and which can result in hypersecretion, in a proportion of which it can be extreme. Whether similar findings may occur in patients with idiopathic GERD treated for prolonged periods (>10 years) with PPIs, at present, is unknown.

Objective: Functional dyspepsia is a common gastrointestinal disorder. The pathogenesis of functional dyspepsia remains unclear. Functional dyspepsia may begin after a bout of gastroenteritis (post-infectious functional dyspepsia) or de novo (nonspecific functional dyspepsia). The aim of this study was to investigate the prevalence and probable mechanisms of post-infectious functional dyspepsia., Material and Methods: Functional dyspepsia patients with a history of unsanitary food intake and acute gastroenteritis 6-12 months ago were enrolled. (13)C-UBT confirmed absence of H. pylori infection. Controls consisted of healthy nondyspeptic volunteers and patients with nonspecific functional dyspepsia. Gastric biopsies were used for routine histology, immunohistochemistry, electron microscopy, ELISA, HPLC assays and Western blot examination., Results: Eighty-five subjects were entered including 35 with post-infectious functional dyspepsia, 30 with nonspecific functional dyspepsia, and 20 healthy controls. The number of mast cells in post-infectious functional dyspepsia and nonspecific functional dyspepsia were significantly greater than that in healthy controls. The number of enterochromaffin cells (ECs) in post-infectious functional dyspepsia was significantly higher than those in nonspecific functional dyspepsia or in healthy controls. The number of mast cells and ECs increased with the density of chronic inflammatory cells. The release of histamine and 5-hydroxytryptamine from gastric mucosa of post-infectious functional dyspepsia patients was significantly greater than those from nonspecific functional dyspepsia or healthy controls. Tryptase protein expression was higher in post-infectious functional dyspepsia and nonspecific functional dyspepsia than in healthy controls. The histological score of chronic gastric inflammation was greater in post-infectious functional dyspepsia versus patients with nonspecific functional dyspepsia or healthy controls. Electron microscopy showed secreting granules in the cytoplasm of both mast cells and ECs. The number of activated mast cells and Ecs at a distance of < 5 microm of nerve fibers were significantly greater in post-infectious functional dyspepsia versus nonspecific functional dyspepsia or controls., Conclusions: Dyspepsia may occur after an acute onset of gastroenteritis in a part of patients. Potent chemicals derived from mast cells and ECs, including histamine, tryptase and 5-hydroxytryptamine may be involved in the pathogenesis of post-infectious functional dyspepsia.

Serotonin (5-HT)-containing enterochromaffin (EC) cells of the intestine transduce chemical and mechanical stimuli from the intestinal lumen by releasing 5-HT on to afferent nerve terminals. Dysfunctional mucosal 5-HT signaling has been implicated in heightened visceral sensitivity and altered motility in patients with inflammatory bowel disease and in animal models. Our aim was to characterize the release and uptake of 5-HT in the mouse dextran sulfate sodium (DSS; 5% wt/vol) model of colitis. We made electrochemical recordings and used an ELISA assay to determine mucosal 5-HT release and uptake in untreated mice and mice with DSS-induced colitis. Peak and steady-state 5-HT concentrations were measured before and during blockade of the serotonin reuptake transporter (SERT) with 1 microM fluoxetine. Electrochemical recordings showed that colons from DSS-treated mice had roughly twice the steady-state levels of extracellular 5-HT and compression-evoked 5-HT release compared with untreated mice. Fluoxetine doubled the compression-evoked and steady-state 5-HT levels in control and DSS mice. These data were supported by ELISA assays, which showed enhanced 5-HT release during colitis, by immunohistochemical analyses, which showed increases in EC cell numbers, and by real-time PCR, which identified a decrease in SERT mRNA expression in the mucosa during colitis. These data are the first to demonstrate 5-HT release close to its release site and near its site of action during DSS-colitis. We conclude that DSS-colitis increases 5-HT availability primarily by an increase in the numbers of EC cells and/or of content of 5-HT in these EC cells.

A generalized absence of enteroendocrine cells characterizes 2 diarrheal/malabsorptive diseases, namely, enteroendocrine cell dysgenesis and autoimmune polyglandular syndrome 1. However, it is not routine for pathologists to examine mucosal biopsies for enteroendocrine cells in cases of chronic diarrheal illness. Our primary aim was to prospectively examine colonic mucosa for loss of enteroendocrine cells using chromogranin A immunohistochemistry for diagnostic purposes. Our secondary aim was to investigate enterochromaffin cells as a subset of enteroendocrine cells, using serotonin (5HT) immunohistochemistry; we hypothesized that other causes of diarrhea due to loss of enteroendocrine cell subsets are missed by evaluating enteroendocrine cells alone. Our approach was limited to patients with chronic unexplained diarrhea partly selected by referring physicians who considered the patients problematic. Seven problematic patients with reduced enteroendocrine or enterochromaffin cells were collected over a 9-month period and placed in group A. Three group A patients demonstrated reduced enteroendocrine cells relative to controls, and they were later diagnosed as having enteroendocrine cell dysgenesis (n = 1) and autoimmune polyglandular syndrome 1 (n = 2). Four group A patients had reduced enterochromaffin cells but normal enteroendocrine cells. These 4 patients had conditions such as congenital diarrhea, mild graft-versus-host disease, acquired childhood chronic diarrhea, and diarrhea post lung transplant. The reduced enterochromaffin cells in the graft-versus-host disease patient inspired a third aim, that is, to investigate whether a loss of enterochromaffin cells would be a generalized defect seen in patients with mild colonic graft-versus-host disease (group B). However, no loss of enterochromaffin cells was detected in group B. Two methods of enumerating endocrine cells were used and demonstrated 67% agreement.

Carcinoids are rare tumors derived from enterochromaffin (EC) cells of the embryonic neural crest. They have malignant potential and their incidence is steadily increasing. The only curative treatment option is surgery. We have focused on cultivation of human neuroendocrine tumors (NET) as relevant models for the study of potential therapy. Only a few cell lines from human carcinoids have been established so far, among them our earlier KRJ-I cell line from a human ileal carcinoid. The reason for the poor success in establishing carcinoid cell lines is due to the small amount of tissue available and the low mitotic activity in primary cultures. We have successfully established three continuously growing cell lines from tissue obtained from a metastatic human carcinoid of the terminal ileum (midgut carcinoid): P-STS was derived from the primary tumor, L-STS from a lymph node metastasis and H-STS from a hepatic metastasis. Immunocytochemistry proved the maintenance of characteristic neuroendocrine properties. Electron microscopy confirmed the presence of neuroendocrine granules. The three cell lines were tumorigenous in SCID-mice. Cytogenetic analyses revealed clonal tetraploidy, inversion and deletion in chromosome 18q, and non-clonal numerical and structural aberrations. Array CGH did not show notable imbalances. Mutation screening of P-STS excluded a MEN1-gene-associated genetic predisposition with high probability. The novel cell lines P-STS, L-STS and H-STS may be useful in vitro and in vivo models for further studies of biological characteristics and the development of new therapeutic agents.

Unlabelled: Enterochromaffin cells (EC) in gastric mucosa produce of serotonin and melatonin and trough its paracrine activity able to change motoric and secretory function. AIM OF OUR STUDIES: To evaluate the density of enterochromaffin cells in gastric mucosa in subjects with functional dyspepsia., Material and Methods: The investigations were performed in 50 subjects including 25 subjects with Postprandial Distress Syndrome and 25 subjects with Epigastric Pain Syndrome accordingly to Rome Criteria III. The comparative group comprised 25 healthy subjects. To identify enterochromaffin cells the bioptates were collected from the corpus and prepyloric part of the stomach and stained immunohistochemically., Results: Number of EC cells in corpus part were--healthy subjects 1.68 +/- 0.35, in patients with PDS--1.26 +/-0.37 (p < 0.001) and in EPS--2.11 +/- 0.43 (p < 0.01). In antrum Number of EC cells were: K--2.52 +/- 0.52, PDS--1.79 +/- 0.3 (p < 0.01), EPS--2.69 +/- 0.46 (p > 0.05). The results of urea breath test (UBT-13C) were: PDS--13.10 +/- 6.49% per hundred, EPS--18.98 +/- 10.11% per hundred (p < 0.05). In group of patients with EPS the positive correlation between number of enterochromaffin cells and urea breath test results was observed., Conclusions: The number of EC cells in gastric mucosa in subjects with functional dyspepsia is different than in healthy subjects. Evaluation of the number of enterochromaffin cells could have clinical value in differential diagnosis in functional dyspepsia.

Objective: To present evidence supporting the hypothesis that the coexistence of gastric carcinoids (GCs) and hyperparathyroidism may represent a distinct clinical entity, not related to multiple endocrine neoplasia type 1 (MEN1)., Methods: We studied a cohort of five young siblings (age range 26-42 years), one of whom had been found to have GC and hyperparathyroidism. All siblings underwent serial gastroscopies for the assessment of gastric neuroendocrine cell proliferations over a mean follow-up period of 31.2 months. Imaging, biochemical and hormonal as well as molecular genetic investigations were performed in the direction of MEN1 syndrome. The literature was searched for cases with coexistence of GCs and hyperparathyroidism not associated with MEN1., Results: Four of the siblings, all male, were found to have GCs in a background of Helicobacter pylori-associated chronic atrophic gastritis and pernicious anaemia, with no serological evidence of gastric autoimmunity. In two of them, asymptomatic hyperparathyroidism was also present. Screening for MEN1 gene mutations or large deletions was negative, and hormone and imaging investigations did not support a diagnosis of familial MEN1 syndrome. A literature search revealed sporadic reports of cases with GC and hyperparathyroidism not attributable to MEN1., Conclusions: The association of GCs and hyperparathyroidism appears to constitute a distinct syndrome that can be encountered in genetically predisposed individuals, and should not be regarded as 'atypical' or 'incomplete' expression of MEN1. Its prevalence and aetiology should be the subject of future studies. Screening for hyperparathyroidism seems to be justified in patients with GC of any type.

This study aimed to investigate the underlying mechanism of protease-activated receptor-2 (PAR-2) agonist-induced visceral hyperalgesia. Male Sprague-Dawley rat pups were submitted to colonic injection of PAR-2 agonist for 6 consecutive days. The visceral sensitivity to colorectal distention was evaluated by electromyography. The enterochromaffin (EC) cell number, 5-HT content and tryrptophan hydroxylase (TPH) protein expression were detected with immunohistochemistry, fluorescent measurement and Western blot analysis. PAR-2 agonist induced a significant increase of visceral nociceptive response to colorectal distention and a series of neurochemical changes in rat colon, including proliferation of EC cells, increased 5-HT content and enhanced TPH expression. Expression of PAR-2 in EC cells was reported for the first time. Further, selective 5-HT(3) receptor antagonist alosteron significantly inhibited PAR-2-induced visceral hyperalgesia. The enhanced 5-HT signaling is likely responsible for the visceral hyperalgesia induced by PAR-2 agonist. Interruption of this pathway is a possible target for the treatment of visceral hyperalgesia in gastrointestinal diseases.

Background/aims: Psychological factors and subtle histopathological changes have been implicated in irritable bowel syndrome (IBS). The aims of the present study were to investigate whether the numbers of enterochromaffin (EC) cells, mast cells, and lamina propria T lymphocytes are altered in IBS, and evaluate the relationship of such alterations with psychological factors., Methods: Forty-two consecutive IBS patients (M : F = 17:25, mean age 48 years) fulfilling the Rome III criteria and twelve asymptomatic healthy controls underwent rectal biopsy. Immunostaining was performed for EC cells, mast cells, and lamina propria T lymphocytes., Results: The IBS group included five post-infectious (PI) IBS and 37 non-PI IBS patients. Significantly more EC cells, mast cells and lamina propria T lymphocytes were observed in PI IBS patients. Mast cells significantly increased in non-PI IBS-D (diarrhea) patients, but not in non-PI IBS-C (constipation) and non-PI IBS-M (mixed) patients. Enterochromaffin cell numbers were not significantly altered in non-PI IBS patients. Anxiety and depression scores did not differ between IBS patients with and without abnormal increase in EC cell or mast cell counts, defined as more than the mean of controls + 2 standard deviations. Enterochromaffin cell, mast cell, or lamina propria T lymphocyte numbers were poorly correlated with anxiety and depression scores in the IBS group., Conclusions: Enterochromaffin cells, mast cells, and lamina propria T lymphocytes significantly increase in PI IBS, whereas only mast cells significantly increase in non-PI IBS-D. Such histopathological changes do not seem to be directly associated with psychological factors.

Background: Gastric carcinoid tumours type 1 (GCA1) originate from hyperplastic enterochromaffin-like (ECL) cells secondary to hypergastrinaemia. Treatment with somatostatin analogues (SSA) might impede ECL-cell hyperplasia by suppressing gastrin secretion and/or by a direct anti-proliferative effect on ECL cells. We conducted a multicentre prospective study to assess the effects of long-acting SSA on hypergastrinaemia and ECL-cell proliferation in patients with GCA1., Methods: We studied 15 patients with GCA1 treated with monthly long-acting release octreotide (LAR) (20-30 mg; n=14) or Lanreotide 90 mg (n=1) for at least 6 months. Patients had serum gastrin and chromogranin A measurements performed and biopsies taken from both tumours and surrounding mucosa before, and every 6-12 months following treatment. Sections were immunostained for neuroendocrine markers. The cell proliferation index Ki-67, intensity of staining before and after treatment and the degree of gastric wall invasion were also assessed., Results: All patients tolerated treatment well (mean follow-up of 18 months). In 11 patients (73%), a complete disappearance of the tumours at 1 year of treatment was observed on endoscopy, while in three patients (20%), the tumours decreased significantly in number and size. Gastrin levels normalized in 25% of patients, and were reduced by more than 80% in the remaining 75%., Conclusions: Treatment with SSAs in GCA1 leads to a substantial tumour load reduction, with a concomitant decrease of serum gastrin levels. Our data indicate an important anti-proliferative effect of SSA on ECL cells, providing clinical benefit and obviating, at least temporarily, the need for invasive therapies for GCA1.

Background/aim: 5-Hydroxytryptamine (5-HT) released from enterochromaffin cells influences intestinal homeostasis by altering gut physiology and is implicated in the pathophysiology of various gut disorders. The mechanisms regulating 5-HT production in the gut remain unclear. This study investigated the T helper (Th) 1/Th2-based immunoregulation of enterochromaffin cell function and 5-HT production in a model of enteric infection., Methods and Results: Trichuris muris-infected AKR (susceptible to infection and generates Th1 response), BALB/c (resistant to infection and generates Th2 response), Stat4-deficient (impaired in Th1 response) and Stat6-deficient (impaired in Th2 response) mice were investigated to assess enterochromaffin cells, 5-HT and cytokines. In association with the generation of a Th2 response we observed higher enterochromaffin cell numbers and 5-HT content in the colon of BALB/c mice compared with AKR mice. Numbers of enterochromaffin cells and amount of 5-HT were significantly lower in Stat6-deficient mice after infection compared with Stat4-deficient mice. In addition, enterochromaffin cell numbers and 5-HT content were significantly higher after reconstitution of severe combined immunodeficient mice with in-vitro polarised Th2 cells., Conclusion: The study demonstrated that enterochromaffin cell and 5-HT responses to the same infectious agent are influenced by Th1 or Th2 cytokine predominance and suggests that the immunological profile of the inflammatory response is important in the regulation of enterochromaffin cell biology in the gut. In addition to new data on enterochromaffin cell function in enteric infection and inflammation, this study provides important information on the immuno-endocrine axis in the gut, which may ultimately lead to improved strategies against gut disorders.

Two cases of gastric tumors showing mixed composition of endocrine cell clusters and exocrine glands and originally diagnosed as mixed neoplasms are described. In both cases, the exocrine glandular component was restricted to the upper third of the neoplasms being consistently absent in areas of muscular wall invasion and, in case 2, in nodal metastases. These glands were in close anatomical contiguity with the glands of the overlying gastric mucosa or, in case 1, apparently derived from deep pouch-like invaginations of the mucosa. They showed either lack of dysplasia (case 1) or mild dysplasia (case 2) with a Ki67 proliferation index consistently lower than that of the intramucosal glands. The intratumoral glands presented intestinal metaplastic features confirmed by intense Cdx2 immunostaining that, conversely, was absent in the endocrine component of the tumors. The latter showed intense vesicular monoamine transporter 2 immunoreactivity consistent with its origin from the enterochromaffin-like cells of the gastric oxyntic mucosa. On the basis of these findings, it is proposed that the exocrine glands do not represent a true neoplastic component of the tumors. Although mucosal entrapment by the tumor cannot be ruled out, they more likely reflect a hitherto unrecognized mechanism of mucosal colonization of gastric endocrine tumors.

Optimal methodological protocol for immunocytochemical demonstration of EC-cells of the rat intestine fixed in 10% neutral buffered formalin with the use of different types of antibodies against serotonin, was developed. It was shown that EC-cells of rat small intestine were better identified with the polyclonal antibodies than with the monoclonal ones. On the contrary, the carcinoid of the human small intestine was better demonstrated with the monoclonal antibodies. In some cases, in mucosal lamina propria mast cells were identified, which were also serotonin-immunoreactive. On the basis of the results obtained, a processing protocol is proposed for the demonstration of EC-cells in the rat gut.

Essential hypertension is a complex disease with both genetic and environmental determinants. The effect of spontaneous hypertension on the distribution and occurrence of somatostatin-, gastrin- and serotonin-immunoreactive cells in the fundus and pylorus of the rat stomach was examined by immunohistochemistry. The animals were killed by decapitation at 4 and 16 weeks of age (5 control rats and 5 hypertensive rats). Endocrine cells generally increase in number in hypertensive rats as compared to control rats. However, the detailed responses of endocrine cells to hypertension depend on the cell type, region of gastric mucosa and age of animals. The present results suggest that hypertension has an influence on the intrinsic regulatory system by endocrine cells control in the rat stomach.

Background: Enterochromaffin (EC) cells are dispersed throughout the gastrointestinal (GI) mucosa and are the main source of 5-hydroxytryptamine (5-HT) in the gut. 5-HT has been implicated in the pathophysiology of several GI disorders, but the mechanisms regulating 5-HT production in the gut are unknown., Aim: To investigate the role of CD4(+) T cells in the production of 5-HT using a model of enteric parasitic infection., Methods and Results: Severe combined immunodeficient (SCID) mice and their wild-type controls were infected with the nematode Trichuris muris and killed on various days after infection to study colonic EC cells and 5-HT production. The number of EC cells and the amount of 5-HT produced were significantly higher in infected wild-type mice than in non-infected mice. The number of EC cells and the amount of 5-HT after infection were significantly lower in SCID mice after infection than in wild-type mice. The number of EC cells and the amount of 5-HT was significantly increased after reconstitution of SCID mice with CD4(+) T cells from infected mice and this was accompanied by an upregulation of colonic CD3 T cells and T helper 2 (Th2) cytokines. Laser capture microdissection-based molecular and immunofluorescence techniques revealed the presence of interleukin 13 receptor alpha1-chain on EC cells., Conclusion: These results show an important immunoendocrine axis in the gut, where secretory products from CD4(+) T cells interact with EC cells to enhance the production of 5-HT in the gut via Th2-based mechanisms. These results show new insights into the mechanisms of gut function, which may ultimately lead to improved therapeutic strategies in functional and inflammatory disorders of the GI tract.

Introduction: Serotonin is an important neuroendocrine transmitter participating in the control of colonic motor activity through neural and biochemical mechanisms in the Enteric Nervous System (ENS). A possible pathophysiological factor for diverticular disease (DD) is altered colonic motility. The study compared the distribution of serotonin cells (SC) in the colonic mucosa of patients with diverticular disease to controls., Methods: Sixteen paraffin specimens with sigmoid diverticular disease were selected and sections of bowel without diverticula from the same specimen were used as its own control. The resection margins from sixteen colonic specimens excised for sigmoid cancer were additional controls. Immunocytochemical staining for serotonin cells was performed on 4-mum tissue sections with polyclonal antibody (NCL-SEROTp). The number of serotonin-positive cells per ten microscopic fields (x200) was assessed in all groups and the staining distribution was defined as low (0-33%), moderate (>33-66%) and high (>66%) according to the percentage of the entire cell containing contrast material. The control specimens were blinded before analysis. Student's t test was used for statistical analysis and significance level was set as P < 0.05., Results: The mean number of serotonin-positive cells per ten fields in the colonic mucosa of specimens with diverticular disease was significantly higher [252.44 (SD 90.64)] than the specimen's own control [147.31 (SD 50.16)] and at normal resection margins of cancer specimens [228.38 (SD 120.10)]. The paired analysis between diverticular disease specimens and its own control (paired t test) showed significant differences for moderate (P = 0.008), high (P = 0.001) and total (P = 0.002) number of serotonin cells. There was no evidence of significance between mean DD and cancer values., Discussion: Increased presence of SCs and the higher proportion of high and moderate staining cells (indicating increased hormone content) indicate the possible role of serotonin in DD. This may be contributing to the pathogenesis of the condition by altered colonic motility in the affected segments in a similar way as in irritable bowel syndrome.

Malignant lymphomas can be first detected in some patients in endoscopic biopsies of the gastrointestinal (GI) tract. However, their recognition and accurate classification often pose problems for the pathologist for several reasons. First, the small sampling size limits pattern recognition and the number of ancillary studies which can be performed. Second, the immune system of the GI tract is capable of intense hyperplastic responses which may mimic lymphoma. Third, in a fashion similar to cutaneous lesions, those in the alimentary tract may be visualized and biopsied at a very early phase in their development when differentiation into neoplasia may be incomplete. Some forms of immune response actually pass through a poorly defined transition into lymphoma. Examples of such 'dysplasia' of the gut immune system include Helicobacter gastritis, coeliac disease and multicentric lymphoid hyperplasia associated with underlying immunodeficiency. With ever increasing endoscopic scrutiny of the gut by gastroenterologists, it is not surprising that the frequency of these indeterminate cases seems to be growing. In combination with careful clinical correlation and conventional microscopic analysis, selective immunohistochemical studies currently constitute the most powerful ancillary method in the pathologist's effort to recognize and classify GI lymphomas accurately.

Mastomys enterochromaffin-like (ECL) cell proliferation is initially gastrin driven, but once neoplasia develops, cells become gastrin autonomous. We hypothesized that CCN2 (CTGF), a mitogenic growth factor, may regulate ECL cell proliferation. A Mastomys GeneChip database was examined (dCHIP) to identify CCN2 expression levels. CCN2 in normal and tumor ECL cell preparations obtained using FACS (100 nM acridine orange) was examined by real-time PCR. CCN2 protein was identified in mucosal and ECL cell preparations by immunohistochemistry. Short-term cultured cells were stimulated with either CCN2 or CCN2 + EGF, and proliferation was measured (MTT assay). The ERK1/2 inhibitor PD-98059 (0.1-100 microM) was assessed in terms of CCN2 (1 ng/ml)-mediated proliferation and ERK1/2 phosphorylation. CCN2 transcript and protein was then examined in clinical gastric carcinoids. The ccn2 transcript was upregulated in tumor samples compared with the normal mucosa (+2.36-fold, P < 0.01). PCR demonstrated that ccn2 was not expressed in FACS-prepared (>98% pure) normal ECL cells but was elevated in tumor ECL cell fractions (41.3 +/- 10.7-fold). Immunostaining of the Mastomys gastric mucosa and FACS preparations confirmed that CCN2 protein was present in ECL tumors but not in normal ECL cells. Neither CCN2 nor CCN2 + EGF stimulated normal ECL cell proliferation. CCN2 stimulated tumor proliferation (EC50 approximately 0.01 ng/ml); EGF significantly augmented (P < 0.01) CCN2-induced tumor cell proliferation (EC50 = 20 pg/ml). PD-98059 inhibited CCN2-induced proliferation (-12 +/- 3%, P < 0.05) and ERK1/2 phosphorylation (-34 +/- 5%, P < 0.05) in tumor cells. In clinical samples, both CCN2 transcript and protein were elevated in gastrin-autonomous carcinoids (P < 0.02) compared with the normal mucosa. In conclusion, CCN2 may be a proliferative regulator of Mastomys ECL neoplastic proliferation once these cells become autonomous of gastrin regulation. Identification of CCN2 in gastric carcinoid tissue may be useful both as an indicator of ECL cell transformation and may define gastrin autonomy, a criteria of gastric carcinoid malignancy.