Cellular localization of guanylin and uroguanylin mRNAs in human and rat duodenal and colonic mucosa.

Guanylin (GUCA2A/Guca2a/GN) and uroguanylin (GUCA2B/Guca2b/UGN) are expressed in the gastrointestinal tract and have been implicated in ion and fluid homeostasis, satiety, abdominal pain, growth and intestinal barrier integrity. Their cellular sources are debated and include goblet cells, entero-/colonocytes, enteroendocrine (EE) cells and tuft cells. We therefore investigated the cellular sources of GN and UGN mRNAs in human and rat duodenal and colonic epithelium with in situ hybridization (ISH) to determine co-expression with Chromogranin A (CHGA/Chga/CgA; enterochromaffin [EC] cells), defensin alpha 6 (DEFA6/Defa6; Paneth cells), mucin 2 (MUC2/Muc2; goblet cells) and selected tuft cell markers. GUCA2A/Guca2a expression was localized to goblet cells and colonocytes in human and rat colon. In human duodenum, GUCA2A was expressed in Paneth cells and was scarce in villous epithelial cells. In rat duodenum, Guca2a was only localized to goblet cells. Guca2b was focally expressed in rat colon. In human and rat duodenum and in human colon, GUCA2B/Guca2b was expressed in dispersed solitary epithelial cells, some with a tuft cell-like appearance. Neither GUCA2A nor GUCA2B were co-expressed with CHGA in human duodenal cells. Consequently, EC cells are probably not the major source of human GN or UGN but other EE cells as a source of GN or UGN are not entirely excluded. No convincing overlap with tuft cell markers was found. For the first time, we demonstrate the cellular expression of GUCA2B in human duodenum. The specific cellular distribution of both GN and UGN differs between duodenum and colon and between human and rat intestines.

The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease.

OBJECTIVE: Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn's disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined. MATERIAL AND METHODS: Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS: GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3',5'-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C. CONCLUSIONS: GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.

The PAS positive material in gastric cancer cells of signet ring type is not mucin.

PURPOSE: The purpose of this study is to assess the exocrine and neuroendocrine properties of tumour cells in diffuse gastric cancer with signet ring cell differentiation. MATERIAL AND METHODS: Mucin mRNA and protein expressions (MUC1, 2, 3, 4, 5AC, 6 and MUC13) were assessed by immunohistochemistry and in situ hybridization. The neuroendocrine properties were evaluated by protein and mRNA expression of the general neuroendocrine markers chromogranin A and synaptophysin. RESULTS: No MUC expression was observed in signet ring tumour cells including the amorphous substance in any of the nine cases. All cases showed immunoreactivity to synaptophysin, and seven out of nine cases immunoreactivity to chromogranin A in signet ring and non-signet ring tumour cells. Chromogranin A mRNA expression was observed in tumour cells in all samples with retained mRNA. CONCLUSIONS: The lack of MUC protein and mRNA in signet ring tumour cells suggests the amorphous substance is not mucin. The lack of MUC mRNA expression in non-signet ring tumour cells questions exocrine differentiation in this tumour group. The abundant protein expression of the general neuroendocrine markers CgA and synaptophysin, and mRNA expression in tumour cells strengthens the hypothesis that this tumour group may be of neuroendocrine origin.

REG gene expression in inflamed and healthy colon mucosa explored by in situ hybridisation.

The regenerating islet-derived (REG) gene family encodes a group of proteins highly expressed in several human pathologies, many of which are associated with epithelial inflammation. All human family members, namely REG1A, REG1B, REG3A and REG4, are closely related in genomic sequence and all are part of the c-type lectin superfamily. REGs are highly expressed during inflammatory bowel disease (IBD)-related colonic inflammation and the in vivo expression pattern of REG1A and REG4 has been localised by using immunohistochemistry. However, the function of the encoded proteins is largely unknown and the cellular localisation of REG expression during colonic inflammation has not been described. Therefore, we have used in situ hybridisation to demonstrate the cellular localisation of REG expression in healthy and diseased colonic mucosa. Samples drawn from an IBD cohort including both inflamed and un-inflamed colonic mucosa are described, as are samples from non-IBD inflammation and healthy controls. Immunohistochemistry against known cell-type markers on serial sections has localised the expression of REGs to metaplastic Paneth cells (REG1A, REG1B and REG3A) and enteroendocrine cells (REG4), with a marked expansion of expression during inflammation. The group of REGs can, based on gene expression patterns, be divided into at least two groups; REG1A, REG1B and REG3A with their expression focused in the crypt base spreading from Paneth cells and REG4 being more highly expressed towards the luminal face. This exploration of expression pattern forms provides the background for further exploration of REG function in the intestine.

Microencapsulation of small intestinal neuroendocrine neoplasm cells for tumor model studies.

Basic cancer research is dependent on reliable in vitro and in vivo tumor models. The serotonin (5-HT) producing small intestinal neuroendocrine tumor cell line KRJ-1 has been used in in vitro proliferation and secretion studies, but its use in in vivo models has been hampered by problems related to the xeno-barrier and tumor formation. This may be overcome by the encapsulation of tumor cells into alginate microspheres, which can function as bioreactors and protect against the host immune system. We used alginate encapsulation of KRJ-1 cells to achieve long-term functionality, growth and survival. Different conditions, including capsule size, variations in M/G content, gelling ions (Ca(2+) /Ba(2+)) and microcapsule core properties, and variations in KRJ-1 cell condition (single cells/spheroids) were tested. Viability and cell growth was evaluated with MTT, and confocal laser scanner microscopy combined with LIVE/DEAD viability stains. 5-HT secretion was measured to determine functionality. Under all conditions, single cell encapsulation proved unfavorable due to gradual cell death, while encapsulation of aggregates/spheroids resulted in surviving, functional bioreactors. The most ideal spheroids for encapsulation were 200-350 µm. Long-term survival (>30 days) was seen with solid Ca(2+) /Ba(2+) microbeads and hollow microcapsules. Basal 5-HT secretion was increased (sixfold) after hollow microcapsule encapsulation, while Ca(2+) /Ba(2+) microbeads was associated with normal basal secretion and responsiveness to cAMP/PKA activation. In conclusion, encapsulation of KRJ-1 cells into hollow microcapsules produces a bioreactor with a high constitutively activate basal 5-HT secretion, while Ca(2+) /Ba(2+) microbeads provide a more stable bioreactor similar to non-encapsulated cells. Alginate microspheres technology can thus be used to tailor different functional bioreactors for both in vitro and in vivo studies.

Differential signal pathway activation and 5-HT function: the role of gut enterochromaffin cells as oxygen sensors.

The chemomechanosensory function of the gut enterochromaffin (EC) cell enables it to respond to dietary agents and mechanical stretch. We hypothesized that the EC cell, which also sensed alterations in luminal or mucosal oxygen level, was physiologically sensitive to fluctuations in O(2). Given that low oxygen levels induce 5-HT production and secretion through a hypoxia inducible factor 1α (HIF-1α)-dependent pathway, we also hypothesized that increasing O(2) would reduce 5-HT production and secretion. Isolated normal EC cells as well as the well-characterized EC cell model KRJ-I were used to examine HIF signaling (luciferase-assays), hypoxia transcriptional response element (HRE)-mediated transcription (PCR), signaling pathways (Western blot), and 5-HT release (ELISA) during exposure to different oxygen levels. Normal EC cells and KRJ-I cells express HIF-1α, and transient transfection with Renilla luciferase under HRE control identified a hypoxia-mediated pathway in these cells. PCR confirmed activation of HIF-downstream targets, GLUT1, IGF2, and VEGF under reduced O(2) levels (0.5%). Reducing O(2) also elevated 5-HT secretion (2-3.2-fold) as well as protein levels of HIF-1α (1.7-3-fold). Increasing O(2) to 100% inhibited HRE-mediated signaling, transcription, reduced 5-HT secretion, and significantly lowered HIF-1α levels (∼75% of control). NF-κB signaling was also elevated during hypoxia (1.2-1.6-fold), but no significant changes were noted in PKA/cAMP. We concluded that gut EC cells are oxygen responsive, and alterations in O(2) levels differentially activate HIF-1α and tryptophan hydroxylase 1, as well as NF-κB signaling. This results in alterations in 5-HT production and secretion and identifies that the chemomechanosensory role of EC cells extends to oxygen sensing.

Management of gastric carcinoids (neuroendocrine neoplasms).

Gastric neuroendocrine neoplasms of the stomach can be divided into the usually well-differentiated, hypergastrinemia-dependent type I and II lesions and the more aggressively behaving gastrin-independent type III lesions. Mainly due to better diagnostics and awareness of this tumor, the observed incidence has increased more than tenfold over the last 30 years. Small (<15-20 mm) localized type I and II lesions that are slowly proliferating (Ki67<2%) can usually be managed conservatively with endoscopic surveillance. Reducing hypergastrinemia by surgical removal of an underlying gastrinoma is important in inhibiting growth and induce reduction of type II lesions, while the specific gastrin receptor antagonist YF476 or gastrin antibodies may become useful for both type I and II lesions. Infiltrating and metastasized tumors and type III lesions require a more aggressive approach with surgical resection and consideration of modalities such as somatostatin analogs, cytotoxics, and peptide receptor targeted treatment.

Parietal cell activation by arborization of ECL cell cytoplasmic projections is likely the mechanism for histamine induced secretion of hydrochloric acid.

BACKGROUND AND AIMS: Enterochromaffin-like (ECL) cells are central in the regulation of acid secretion. G cells release gastrin and activate ECL cell histamine secretion which stimulates parietal cell H(2) receptors initiating acid secretion. It is unclear whether histamine-mediated parietal cell activation is via a vascular or paracrine pathway. To assess this, we utilized immunohistochemistry (IHC) and electron microscopy to examine gastric tissue and used visualization of formalin fixed dispersed gastric cells and glands to investigate and define the anatomical relationship between ECL and parietal cells. MATERIAL AND METHODS: Sprague-Dawley rat stomachs were instilled with formalin. Thereafter fixed mucosal cells and whole gastric glands were dispersed by mechanical and chemical dissolution and enzymatic digestion. Smears with fixed isolated cells and whole glands were stained by IHC with histidine decarboxylase (HDC) and H+/K+-ATPase antibodies. Whole tissue samples of Sprague-Dawley and cotton rat oxyntic mucosa were investigated with IHC using HDC, VMAT2 and H+/K+-ATPase antibodies, and electron microscopy was performed to further delineate the precise anatomic relationship between ECL cells and parietal cells. RESULTS: Each ECL cell generated a network of HDC- and VMAT2-positive dendritic-like elongations that were in direct contact with several parietal cells. Thus, ECL cells at the base of the gland were in communication with parietal cells in the middle of the gland. Electron microscopy confirmed that the cytoplasmic ECL cell elongations containing secretory vesicles were in direct juxtaposition to parietal cells. CONCLUSIONS: These findings indicate that ECL cells directly regulate parietal cell function in a neurocrine manner via slender neuron-like elongations.

The peroxisome proliferator-activated receptor (PPAR) alpha agonist fenofibrate maintains bone mass, while the PPAR gamma agonist pioglitazone exaggerates bone loss, in ovariectomized rats.

BACKGROUND: Activation of peroxisome proliferator-activated receptor (PPAR)gamma is associated with bone loss and increased fracture risk, while PPARalpha activation seems to have positive skeletal effects. To further explore these effects we have examined the effect of the PPARalpha agonists fenofibrate and Wyeth 14643, and the PPARgamma agonist pioglitazone, on bone mineral density (BMD), bone architecture and biomechanical strength in ovariectomized rats. METHODS: Fifty-five female Sprague-Dawley rats were assigned to five groups. One group was sham-operated and given vehicle (methylcellulose), the other groups were ovariectomized and given vehicle, fenofibrate, Wyeth 14643 and pioglitazone, respectively, daily for four months. Whole body and femoral BMD were measured by dual X-ray absorptiometry (DXA), and biomechanical testing of femurs, and micro-computed tomography (microCT) of the femoral shaft and head, were performed. RESULTS: Whole body and femoral BMD were significantly higher in sham controls and ovariectomized animals given fenofibrate, compared to ovariectomized controls. Ovariectomized rats given Wyeth 14643, maintained whole body BMD at sham levels, while rats on pioglitazone had lower whole body and femoral BMD, impaired bone quality and less mechanical strength compared to sham and ovariectomized controls. In contrast, cortical volume, trabecular bone volume and thickness, and endocortical volume were maintained at sham levels in rats given fenofibrate. CONCLUSIONS: The PPARalpha agonist fenofibrate, and to a lesser extent the PPARaplha agonist Wyeth 14643, maintained BMD and bone architecture at sham levels, while the PPARgamma agonist pioglitazone exaggerated bone loss and negatively affected bone architecture, in ovariectomized rats.

The archaic distinction between functioning and nonfunctioning neuroendocrine neoplasms is no longer clinically relevant.

BACKGROUND: Neuroendocrine neoplasms (NENs) are increasing in incidence and prevalence. This reflects greater clinical awareness, effective imaging, and increasing pathological diagnostic recognition. Although the identification and treatment of clinical neuroendocrine syndromes are established, there is confusion when a NEN has no discernible clinical symptoms. DISCUSSION: Nonfunctional tumors are usually diagnosed incidentally and at a later stage largely because either they do not secrete a bioactive product or do so, but in a form that is either inactive or in quantities that have no discernible effect. Nevertheless, the histopathology is indistinguishable from functional NENs, and tumors exhibit somatostatin receptor expression, and positive immunohistochemistry for neuroendocrine cell markers (CgA, NSE/synaptophysin). Similarly, their rates of growth and metastatic behavior are, like other NENs, predictably based on staging and grading (mitotic rate and Ki67 expression). Both types are diagnosed biochemically (CgA) and by imaging in an identical fashion with computed tomography, magnetic resonance imaging, somatostatin receptor scintigraphy, and endoscopic ultrasound. NENs, irrespective of function or bioactive secretory profile, respond with equal efficacy to the same regimen of surgery or antitumor drugs (e.g., somatostatin analogs with or without tyrosine kinase inhibitors/antiangiogenics or cytotoxics) depending on grade. Given the efficacy of somatostatin analogs in increasing progression free survival, nonfunctional NENs should be managed identically to symptomatic NENs. The consideration of NENs as functional or nonfunctional is an archaic clinical concept that should be discarded since the tumors are indistinguishable at a cellular, biological, and morphological level. All current evidences indicate that their diagnosis and treatment should follow the same common principles.

Delineation of the chemomechanosensory regulation of gastrin secretion using pure rodent G cells.

BACKGROUND & AIMS: Gastrin is a key regulator of gastric acid secretion. We aimed to isolate pure G cells to identify the mechanistic basis of luminal- and strain-mediated regulation. METHODS: Using gradient centrifugation and fluorescence-activated cell sorting, rat G cells were prepared and luminal, neural, hormonal, and mechanical activation of secretion and signaling pathways studied. RESULTS: Pure G-cell preparations (>97%) were isolated. Reverse-transcription polymerase chain reaction identified neural, hormonal, bacterial, and luminal G protein-coupled receptors, and immunostaining visualized specific sweet/bitter receptors and the tastant-associated G protein alpha-gustducin. Gastrin release was stimulated by forskolin (adenosine 3',5'-cyclic monophosphate [cAMP] inducer, 10 micromol/L; >3-fold), potentiated by 3-isobutyl-1-methylxanthine (IBMX; phosphodiesterase type 5 inhibitor and adenosine antagonist, 10 micromol/L) and phorbol myristate acetate (phorbol ester, 10 micromol/L), and inhibited by H-89 (protein kinase A inhibitor, 10 micromol/L), PD98059 (MEK1 inhibitor, 0.1 micromol/L), and wortmannin (phosphatidylinositol 3-kinase inhibitor, 1 nmol/L). Gastrin release was stimulated by neuronal G protein-coupled receptor ligands, pituitary adenylate cyclase-activating protein (20 pmol/L, >8-fold) and bombesin (0.1 micromol/L, 8-fold) through cAMP signaling. The tastants sucralose, glucose, caffeine, denatonium, and the vanilloid receptor activator capsaicin all stimulated secretion (>3-fold), as did bacterial lipopolysaccharides Salmonella enteritidis (0.24 nmol/L, 5-fold) greater than Helicobacter pylori (0.57 micromol/L, 3-fold). Secretion was associated with elevated cAMP levels (approximately 2-fold) and could be inhibited by H-89 and PD98059 and potentiated by IBMX and cholera toxin (250 microg/mL). Bacterially mediated secretion also involved activation of nuclear factor kappaB and the c-Jun-N-terminal kinase pathway. Mechanical strain stimulated (2-fold to 8-fold) gastrin release, and decreasing pH from 7.4 to 5.5 inhibited release. The adenosine receptor 2B antagonist MRS1754 inhibited mechanically induced gastrin release. CONCLUSIONS: G cells are luminal sampling chemomechanosensory cells whose secretion is regulated by neural, hormonal, luminal, and mechanical factors through protein kinase A activation, cAMP signaling, and mitogen-activated protein kinase phosphorylation.

Chromogranin A--biological function and clinical utility in neuro endocrine tumor disease.

BACKGROUND: Neuroendocrine tumors (NETs) are a form of cancer that differ from other neoplasia in that they synthesize, store, and secrete peptides, e.g., chromogranin A (CgA) and amines. A critical issue is late diagnosis due to failure to identify symptoms or to establish the biochemical diagnosis. We review here the utility of CgA measurement in NETs and describe its biological role and the clinical value of its measurement. METHODS: Literature review and analysis of the utility of plasma/serum CgA measurements in NETs and other diseases. RESULTS: CgA is a member of the chromogranin family; its transcription and peptide processing are well characterized, but its precise function remains unknown. Levels are detectable in the circulation but vary substantially (approximately 25%) depending on which assay is used. Serum and plasma measurements are concordant. CgA is elevated in approximately 90% of gut NETs and correlates with tumor burden and recurrence. Highest values are noted in ileal NETs and gastrointestinal NETs associated with multiple endocrine neoplasia type 1. Both functioning and nonfunctioning pancreatic NETs have elevated values. CgA is more frequently elevated in well-differentiated tumors compared to poorly differentiated NETs. Effective treatment is often associated with decrease in CgA levels. Proton pump inhibitors falsely increase CgA, but levels normalize with therapy cessation. CONCLUSIONS: CgA is currently the best available biomarker for the diagnosis of NETs. It is critical to establish diagnosis and has some utility in predicting disease recurrence, outcome, and efficacy of therapy. Measurement of plasma CgA is mandatory for the effective diagnosis and management of NET disease.

A nomogram to assess small-intestinal neuroendocrine tumor ('carcinoid') survival.

Neuroendocrine tumors (NETs) are a heterogeneous group of cancers of which the commonest site is the small intestine (SI). Most information available to determine tumor behavior reflects univariate assessment of factors or is anecdotal or experience based. There currently exists no objective multivariate analysis of indices that defines SI NET prognosis. A key unmet need is the lack of a rigorous mathematical-based tool - a nomogram - for the assessment of parameters that define progress, determine prognosis and can guide therapy. Since prediction of NET behavior is a critical criterion in determining clinical strategy, we constructed a NET nomogram (Modlin Score) for prognosis prediction, patient group comparisons and a guide for stratification of treatment and surveillance. We used hazard ratio (HR), Cox analysis and Kaplan-Meier analysis of published data and the current Surveillance, Epidemiology and End Results (SEER) database (approx. 20,000 patients) to develop a nomogram from 15 variables demonstrated to provide significant multivariate HRs. These included age, gender, ethnicity, symptoms, urinary 5-hydroxyindoleacetic acid, plasma chromogranin A, liver function tests, tumor size, invasion, metastasis, histology, Ki-67 index, carcinoid heart disease and therapy (surgery or long-acting somatostatin analogs). Internal validation was assessed using 33 SI NET patients. A NET nomoscore (Modlin Score) was developed by HR weighting and stratification into low (<75), medium (75-95) and high risk (>95). This identified significant differences (p <0.03, Kaplan-Meier) in survival (15.5 ± 4.3, 9.7 ± 2.5 and 6.4 ± 1.1 years, respectively). The Modlin Score was significantly elevated (p <0.01) in deceased compared to alive patients. This nomogram represents an optimized construct based upon currently analyzable data, and application will facilitate accurate stratification for comparison in clinical trials. External validation and amplification by identification of additional indices, e.g. molecular biomarkers, are necessary. The development of a mathematically validated nomogram provides a platform for objective assessment of SI NET disease, a finite basis for precise prognostication and a tool to guide management strategy.

Neuroendocrine tumors of the gallbladder: an evaluation and reassessment of management strategy.

BACKGROUND: Gallbladder neuroendocrine tumors (GB-NETs) represent only 0.5% of all NETs, and little is known about their biological behavior. We sought to provide an overview of the current state of knowledge about GB-NETs and provide a recommendation for management. STUDY: A PubMed search was undertaken using the following criteria: primary gallbladder and carcinoid or NET. We also interrogated the SEER 9, 13 and 17 registries (1973 to 2005) and the Niigata registry (2003) to assess the epidemiology and clinicopathological characteristics. Finally, we compared the clinical presentation, management and prognosis of GB-NETs to that of gallbladder adenocarcinoma. RESULTS: GB-NETs probably derive from either a multipotent stem cell or neuroendocrine cells in intestinal or gastric metaplasia of the gallbladder epithelium, which occurs consequent upon cholelithiasis/chronic inflammation. Clinically and at surgery, GB-NETs are indistinguishable from gallbladder cancer (GBC) and "carcinoid syndrome" is evident in only ∼1%.The median survival was only 9.8 months among 278 cases of GB-NETs reported in SEER. The 5 year survival rate for tumors classified as carcinoids/neuroendocrine carcinoma or small cell cancer (SCC) was 36.9 and 0%, respectively. Soga divided GB-NETs into typical and variant carcinoids with 5 year survival rates of 60.4% and 21.3% respectively. CONCLUSIONS: GB-NETs have an aggressive behavior, and once diagnosed, extensive surgical management and careful NET follow up with CT scan is mandatory to facilitate early detection of recurrence. Since more aggressive surgical management for GBC has shown increased survival rates for these tumors, a similar strategy seems reasonable for GB-NETs. However, in high grade metastatic tumors, the primary management is mainly medical.

Serotonin and fluoxetine receptors are expressed in enamel organs and LS8 cells and modulate gene expression in LS8 cells.

The selective serotonin re-uptake inhibitor (SSRI) fluoxetine is widely used in the treatment of depression in children and fertile women, but its effect on developing tissues has been sparsely investigated. The aim of this study was to investigate if enamel organs and ameloblast-derived cells express serotonin receptors that are affected by peripherally circulating serotonin or fluoxetine. Using RT-PCR and western blot analysis we found that enamel organs from 3-d-old mice and ameloblast-like cells (LS8 cells) express functional serotonin receptors, the rate-limiting enzyme in serotonin synthesis (Thp1), as well as the serotonin transporter (5HTT), indicating that enamel organs and ameloblasts are able to respond to serotonin and regulate serotonin availability. Fluoxetine and serotonin enhanced the alkaline phosphatase activity in the cell culture medium from cultured LS8 cells, whereas the expression of enamelin (Enam), amelogenin (Amel), and matrix metalloproteinase-20 (MMP-20) were all significantly down-regulated. The secretion of vascular endothelial growth factor (VEGF), monocyte chemotactic protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10) was also reduced compared with controls. In conclusion, enamel organs and ameloblast-like cells express functional serotonin receptors. Reduced transcription of enamel proteins and secretion of vascular factors may indicate possible adverse effects of fluoxetine on amelogenesis.

Principal component analysis, hierarchical clustering, and decision tree assessment of plasma mRNA and hormone levels as an early detection strategy for small intestinal neuroendocrine (carcinoid) tumors.

Incidence of neuroendocrine tumors (NETs) is increasing (approximately 6%/year), but clinical presentation is nonspecific, resulting in delays in diagnosis (5-7 years; approximately 70% have metastases). This reflects absence of a sensitive plasma marker. The aim of this study is to investigate whether detection of circulating messenger RNA (mRNA) alone or in combination with circulating NET-related hormones and growth factors can detect gastrointestinal NET disease. The small intestinal (SI) NET cell line KRJ-I was used to define the sensitivity of real-time polymerase chain reaction (PCR) for mRNA detection in blood. NSE, Tph-1, and VMAT ( 2 ) transcripts were identified from one KRJ-I cell/ml blood. mRNA from the tissue and plasma of SI-NETs (n = 12) and gastric NETs (n = 7), and plasma from healthy controls (n = 9) was isolated and real-time PCR performed. Tph-1 was a specific marker of SI-NETs (58%, p < 0.03) whereas CgA transcripts did not differentiate tumors from controls. Patients with metastatic disease expressed more marker transcripts than localized tumors (75% versus 18%, p < 0.02). Plasma 5-hydroxytryptamine (5-HT), chromogranin A (CgA), ghrelin, and connective tissue growth factor (CTGF) fragments were measured, combined with mRNA levels, and a predictive mathematical model for NET diagnosis developed using decision trees. The sensitivity and specificity to diagnose SI-NETs and gastric NETs were 81.2% and 100%, and 71.4% and 55.6%, respectively. We conclude that mRNA from one NET cell/ml blood can be detected. Circulating plasma Tph-1 is a promising marker gene for SI-NET disease (specificity 100%) while an increased number of marker transcripts (>2) correlated with disease spread. Including NET-related circulating hormones and growth factors in the algorithm increased the sensitivity of detection of SI-NETs from 58 to 82%.

KRJ-I and BON cell lines: defining an appropriate enterochromaffin cell neuroendocrine tumor model.

BACKGROUND: Neuroendocrine tumors (NETs) of the gastrointestinal (GI) system are increasing in incidence with minimal improvement in prognosis. Although the cell of origin has been identified as the enterochromaffin (EC) cell, its secretory and proliferative regulation has not been defined at a mechanistic level. To date, the BON cell line has been the most widely used in vitro EC cell model despite its pancreatic origin. Using whole-genome mathematical analysis as well as secretory and proliferative studies, we compared the BON cell line to the small intestine (SI) EC cell-derived NET cell line, KRJ-I, to assess individual cell line validity and applicability for the investigation of GI-NET disease. METHODS AND RESULTS: Principal component analysis and ANOVA of KRJ-I and BON transcriptomes (U133 Plus 2) identified substantially different (<10%) overlap in transcripts with minimal (R(2) = 0.24) correlation in gene expression profiles. RT-PCR detected large variability (>12%) in neuroendocrine (NE) marker transcripts in the BON cell line and the absence of Tph-2, DDC, TGFbetaR2, and M3 transcripts in KRJ-I. The KRJ-I cell line secreted serotonin (5-HT) in response to isoproterenol (EC(50) = 100 nM), noradrenaline (EC(50) = 1.7 nM), and pituitary adenylate cyclase (PACAP, EC(50) = 0.03 nM). Cholecystokinin (IC(50) = 430 nM), somatostatin (IC(50) = 400 nM), acetylcholine (IC(50) = 3.7 nM), and gamma-aminobutyric acid A (GABA(A), IC(50) = 2 nM) all inhibited 5-HT release, while gastrin and bombesin had no effect. 5-HT secretion in the BON cell line was stimulated by isoproterenol (EC(50) = 900 nM), noradrenaline (EC(50) = 20 nM), cholecystokinin (EC(50) = 130 nM), PACAP (EC(50) = 0.12 nM), bombesin (EC(50) = 15 nM), and acetylcholine (EC(50) = 0.2 nM). It was inhibited by somatostatin (IC(50) = 300 nM) but not GABA(A). KRJ-I responded with proliferation to connective tissue growth factor (CTGF, EC(50) = 0.002 ng/ml), transforming growth factor-alpha (TGFalpha, EC(50) = 0.63 ng/ml) and transforming growth factor-beta (TGFbeta, EC(50) = 0.63 ng/ml). Epidermal growth factor (EGF) and somatostatin had no significant effect. BON cell proliferation was stimulated only by EGF and TGFalpha (EC(50) = 15.8 and 10 ng/ml). TGFbeta (IC(50) = 0.16 ng/ml), MZ-4-147 (IC(50) = 0.5 nM), and BIM23A761 (IC(50) = 0.06 nM) all inhibited proliferation. CTGF and somatostatin had no effect. CONCLUSION: KRJ-I and BON cell lines demonstrate substantial differences in gene level transcripts, inconsistent receptor profile expression, wide variability in NE marker transcript levels, and significantly differential proliferative and secretory responses. Given the EC cell origin of KRJ-I, these results provide evidence that the BON cell line does not represent an EC cell system and is not a valid study model of (carcinoid) EC cell-derived NET.

Different skeletal effects of the peroxisome proliferator activated receptor (PPAR)alpha agonist fenofibrate and the PPARgamma agonist pioglitazone.

BACKGROUND: All the peroxisome proliferator activated receptors (PPARs) are found to be expressed in bone cells. The PPARgamma agonist rosiglitazone has been shown to decrease bone mass in mice and thiazolidinediones (TZDs) have recently been found to increase bone loss and fracture risk in humans treated for type 2 diabetes mellitus. The aim of the study was to examine the effect of the PPARalpha agonist fenofibrate (FENO) and the PPARgamma agonist pioglitazone (PIO) on bone in intact female rats. METHODS: Rats were given methylcellulose (vehicle), fenofibrate or pioglitazone (35 mg/kg body weight/day) by gavage for 4 months. BMC, BMD, and body composition were measured by DXA. Histomorphometry and biomechanical testing of excised femurs were performed. Effects of the compounds on bone cells were studied. RESULTS: The FENO group had higher femoral BMD and smaller medullary area at the distal femur; while trabecular bone volume was similar to controls. Whole body BMD, BMC, and trabecular bone volume were lower, while medullary area was increased in PIO rats compared to controls. Ultimate bending moment and energy absorption of the femoral shafts were reduced in the PIO group, while similar to controls in the FENO group. Plasma osteocalcin was higher in the FENO group than in the other groups. FENO stimulated proliferation and differentiation of, and OPG release from, the preosteoblast cell line MC3T3-E1. CONCLUSION: We show opposite skeletal effects of PPARalpha and gamma agonists in intact female rats. FENO resulted in significantly higher femoral BMD and lower medullary area, while PIO induced bone loss and impairment of the mechanical strength. This represents a novel effect of PPARalpha activation.

Uncommon cancers of the small intestine, appendix and colon: an analysis of SEER 1973-2004, and current diagnosis and therapy.

Analysis of the Surveillance, Epidemiology and End Results (SEER) registry indicates that more than 20% of all cancers are located in the gastrointestinal (GI) tract. Although colon adenocarcinomas constitute approximately 90% of all malignant intestinal neoplasia, the remaining 10% of tumors in the small intestine (SI), appendix and colon are clinically relevant since their late presentation due to a paucity of overt symptoms culminates in a high mortality rate despite the fact that many such lesions are not intrinsically aggressive neoplasia. Thus, neuroendocrine tumors (NETs), adenocarcinomas (except for colonic), lymphomas, sarcomas and GI stromal tumors (GISTs) of the SI, appendix and colon, while relatively rare, represent an under-recognized and underserved group of lesions. According to the SEER registry 1973-2004, the incidence/100,000 of sarcomas has remained unchanged, while NETs, adenocarcinomas (except colon), and lymphomas have increased 2.9-, 1.6-, and 2.0-fold, respectively. This may, at least partly, reflect the development of more sophisticated diagnostic techniques including high resolution CT and MRI, capsule endoscopy and somatostatin scintigraphy for NETs. Although the development of specific targeted therapies such as tyrosine kinase inhibitors (TKIs) for GISTs and somatostatin analogs for NETs have improved prognosis, early detection remains the critical variable in determining outcome. The overall 5-year survival rates have remained relatively unchanged over time (1973-1999), or are only improved marginally for some subgroups. We present an overview of the epidemiology of these uncommon cancers, and address their clinical behavior, and current diagnostic and therapeutic options.

The effect of terguride in carbon tetrachloride-induced liver fibrosis in rat.

BACKGROUND: Hepatic stellate cells (HSCs) are considered to be of vital importance in the pathogenesis of liver fibrosis. In vitro studies have demonstrated antiproliferative effects of 5-hydroxytryptamine(2) (5-HT(2), serotonin) receptor antagonists upon HSCs. Terguride, recognized as a partial dopamine agonist, also has potent 5-HT(2) receptor antagonist qualities and has been shown to prevent serotonin-induced organ changes and fibrosis in rats. AIM: In the current study, the carbon tetrachloride (CCL(4)) hepatic fibrosis rat model was used in combination with daily administration of terguride to evaluate potential preventive effects of a 5-HT(2) receptor antagonist upon liver fibrosis. MATERIALS AND METHODS: Forty rats (Sprague-Dawley) were included in the study. Twelve animals received terguride in combination with CCL(4) and 12 animals were given only CCL(4). The remaining 16 animals served as model controls. The extent of fibrosis was evaluated by liver weight, histologic analysis, biochemical analysis, and alpha-smooth muscle actin (alpha-SMA) immunohistochemistry. RESULTS: There were no significant differences in liver weight, hydroxyproline content, serum alanine and aspartate transaminases, serum hyaluronic acid, or alpha-SMA immunostaining between rats treated with terguride in combination with CCL(4) and rats given only CCL(4). All parameters, however, were significantly lower (P < 0.01) in the model controls. CONCLUSION: Terguride, a potent 5-HT(2) antagonist, did not prevent the development of liver fibrosis in rats given CCL(4).