Alterations in mucosal immunity identified in the colon of patients with irritable bowel syndrome.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) has been associated with mucosal dysfunction, mild inflammation, and altered colonic bacteria. We used microarray expression profiling of sigmoid colon mucosa to assess whether there are stably expressed sets of genes that suggest there are objective molecular biomarkers associated with IBS. METHODS: Gene expression profiling was performed using Human Genome U133 Plus 2.0 (Affymetrix) GeneChips with RNA from sigmoid colon mucosal biopsy specimens from 36 IBS patients and 25 healthy control subjects. Real-time quantitative polymerase chain reaction was used to confirm the data in 12 genes of interest. Statistical methods for microarray data were applied to search for differentially expressed genes, and to assess the stability of molecular signatures in IBS patients. RESULTS: Mucosal gene expression profiles were consistent across different sites within the sigmoid colon and were stable on repeat biopsy over approximately 3 months. Differentially expressed genes suggest functional alterations of several components of the host mucosal immune response to microbial pathogens. The most strikingly increased expression involved a yet uncharacterized gene, DKFZP564O0823. Identified specific genes suggest the hypothesis that molecular signatures may enable distinction of a subset of IBS patients from healthy controls. By using 75% of the biopsy specimens as a validation set to develop a gene profile, the test set (25%) was predicted correctly with approximately 70% accuracy. CONCLUSIONS: Mucosal gene expression analysis shows there are relatively stable alterations in colonic mucosal immunity in IBS. These molecular alterations provide the basis to test the hypothesis that objective biomarkers may be identified in IBS and enhance understanding of the disease.

Molecular profiling of murine sensory neurons in the nodose and dorsal root ganglia labeled from the peritoneal cavity.

Vagal afferent neurons are thought to convey primarily physiological information, whereas spinal afferents transmit noxious signals from the viscera to the central nervous system. To elucidate molecular identities for these different properties, we compared gene expression profiles of neurons located in nodose ganglia (NG) and dorsal root ganglia (DRG) in mice. Intraperitoneal administration of Alexa Fluor-488-conjugated cholera toxin B allowed enrichment for neurons projecting to the viscera. Fluorescent neurons in DRG (from T10 to T13) and NG were isolated using laser-capture microdissection. Gene expression profiles of these afferent neurons, obtained by microarray hybridization, were analyzed using multivariate spectral map analysis, significance analysis of microarrays (SAM) algorithm, and fold-difference filtering. A total of 1,996 genes were differentially expressed in DRG vs. NG, including 41 G protein-coupled receptors and 60 ion channels. Expression profiles obtained on laser-captured neurons were contrasted to those obtained on whole ganglia, demonstrating striking differences and the need for microdissection when studying visceral sensory neurons because of dilution of the signal by somatic sensory neurons. Furthermore, we provide a detailed catalog of all adrenergic and cholinergic, GABA, glutamate, serotonin, and dopamine receptors; voltage-gated potassium, sodium, and calcium channels; and transient receptor potential cation channels present in afferents projecting to the peritoneal cavity. Our genome-wide expression profiling data provide novel insight into molecular signatures that underlie both functional differences and similarities between NG and DRG sensory neurons. Moreover, these findings will offer novel insight into mode of action of pharmacological agents modulating visceral sensation.

AG013, a mouth rinse formulation of Lactococcus lactis secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis.

Non-clinical studies, focusing on the pharmacodynamics (PD), pharmacokinetics (PK) and safety pharmacology of genetically modified Lactococcus lactis (L. lactis) bacteria, engineered to secrete human Trefoil Factor 1 (hTFF1), were performed to provide proof-of-concept for the treatment of oral mucositis (OM) patients. L. lactis strain sAGX0085 was constructed by stably inserting an htff1 expression cassette into the bacterial genome, and clinically formulated as a mouth rinse (coded AG013). PD studies, using different oral dosing regimens, were performed in a clinically relevant hamster model for radiation-induced OM. The PK profile was assessed in healthy hamsters and in hamsters with radiation-induced OM. In addition, in vitro and in vivo safety pharmacology studies were conducted, in pooled, complement-preserved human serum, and in neutropenic hamsters and rats respectively. Topical administration of L. lactis sAGX0085/AG013 to the oral mucosa significantly reduced the severity and course of radiation-induced OM. PK studies demonstrated that both living L. lactis bacteria, as well as the hTFF1 secreted, could be recovered from the administration site for maximum 24h post-dosing, without systemic exposure. The in vitro and in vivo safety pharmacology studies confirmed that L. lactis sAGX0085 could not survive in systemic circulation, not even under neutropenic conditions. The results from the PD, PK and safety pharmacology studies reported here indicate that in situ secretion of hTFF1 by topically administered L. lactis bacteria provides a safe and efficacious therapeutic tool for the prevention and treatment of OM.

Actobiotics as a novel method for cytokine delivery.

Interleukin-10 (IL-10) is central in immune downregulation, but so far its use in inflammatory diseases remains cumbersome. For treatment of inflammatory bowel disease, adequate amounts of IL-10 must reach the intestinal lining. Systemic injection of a pharmacologically active doses of recombinant human (rh) IL-10 results in very low mucosal levels of protein and severe toxicity and side effects. In animal models, topical and active delivery of IL-10 by ingestion of recombinant Lactococcus lactis (L. lactis) was shown to be a valuable alternative. Starting thereof we have developed a novel pharmaceutical platform. Our expertise and TopAct (topical and active) delivery technology allows use of recombinant L. lactis- ActoBiotics- in clinical practice. Here we discuss the development of recombinant L. lactis for intestinal delivery of rhIL-10 in humans.

The hepatocarcinoma cell line HepG2 does not express a GHS-R1a-type ghrelin receptor.

The interaction of ghrelin, a 28-residue acylated peptide, with the growth hormone secretagogue receptor 1a (GHS-R1a) has been studied mostly in cells expressing a recombinant GHS-R1a. As awareness is growing on the importance to study G protein-coupled receptors in a natural environment, we studied the effect of ghrelin in the human hepatocellular HepG2 cell line because it has been described in literature to respond to ghrelin. Despite extensive efforts, we were not able to confirm mRNA expression of GHS-R1a by reverse transcription PCR, radioligand binding, or ghrelin-induced GHS-R1a receptor activation; therefore, we conclude that HepG2 cells do not express GHS-R1a. On the other hand, we confirmed a modest effect of ghrelin on the up-regulation of IRS-1 phosphorylation, which might suggest the existence of an alternative ghrelin receptor in HepG2 cells.

Alterations in the brain-gut axis underlying visceral chemosensitivity in Nippostrongylus brasiliensis-infected mice.

BACKGROUND & AIMS: Visceral hypersensitivity, a hallmark of irritable bowel syndrome, is generally considered to be mechanosensitive in nature and mediated via spinal afferents. Both stress and inflammation are implicated in visceral hypersensitivity, but the underlying molecular mechanisms of visceral hypersensitivity are unknown. METHODS: Mice were infected with Nippostrongylus brasiliensis (Nb) larvae, exposed to environmental stress and the following separate studies performed 3-4 weeks later. Mesenteric afferent nerve activity was recorded in response to either ramp balloon distention (60 mm Hg), or to an intraluminal perfusion of hydrochloric acid (50 mmol/L), or to octreotide administration (2 micromol/L). Intraperitoneal injection of cholera toxin B-488 identified neurons projecting to the abdominal viscera. Fluorescent neurons in dorsal root and nodose ganglia were isolated using laser-capture microdissection. RNA was hybridized to Affymetrix Mouse whole genome arrays for analysis to evaluate the effects of stress and infection. RESULTS: In mice previously infected with Nb, there was no change in intestinal afferent mechanosensitivity, but there was an increase in chemosensitive responses to intraluminal hydrochloric acid when compared with control animals. Gene expression profiles in vagal but not spinal visceral sensory neurons were significantly altered in stressed Nb-infected mice. Decreased afferent responses to somatostatin receptor 2 stimulation correlated with lower expression of vagal somatostatin receptor 2 in stressed Nb-infected mice, confirming a link between molecular data and functional sequelae. CONCLUSIONS: Alterations in the intestinal brain-gut axis, in chemosensitivity but not mechanosensitivity, and through vagal rather than spinal pathways, are implicated in stress-induced postinflammatory visceral hypersensitivity.

Alterations in expression of p11 and SERT in mucosal biopsy specimens of patients with irritable bowel syndrome.

BACKGROUND & AIMS: The pathophysiology of irritable bowel syndrome (IBS) remains enigmatic; abnormalities in serotonin metabolism have been implicated. Two proteins that influence the function of serotonin and serotonergic receptors are serotonin transporter protein (SERT or soluble carrier protein, SLC6A4) and p11 (S-100A10, or calpactin I light chain). Both proteins are reported to be associated with depression-like states, a frequent comorbid condition in IBS. We explored the hypothesis that expression of these 2 proteins in colonic and rectal mucosa is abnormal in patients with IBS as compared with healthy controls. METHODS: Messenger RNA (mRNA) expression of SLC6A4 and p11 was measured in sigmoid and rectal mucosal biopsy specimens. Genotype of the promoter for SLC6A4 was also assessed in all participants. Validation studies explored reproducibility of 2 biopsy specimens taken from the same region and biopsy specimens taken an average of approximately 3 months apart. RESULTS: We found normal colonic mucosal expression of SLC6A4 in diarrhea (IBS-D)- or constipation-predominant IBS (IBS-C). On the other hand, p11 expression was increased in IBS. No significant effect on p11 mRNA expression in sigmoid colon or rectum was noted from antidepressant treatment in any of the analyzed subgroups. CONCLUSIONS: Colonic mucosal expression of SLC6A4 in IBS is normal. Given that overexpression of p11 can increase serotonergic receptor functions (eg, 5-HT(1B) receptors), these data support the need for further study of the interaction between p11 expression in health and disease and its role in the therapeutic response to serotonergic agents, including antidepressants.

Influence of motilin on gastric fundus tone and on meal-induced satiety in man: role of cholinergic pathways.

BACKGROUND: Motilin agonists are strong gastroprokinetics, but their impact on symptoms in delayed gastric emptying has been disappointing. It has been speculated that it is due to the contractile effect of motilin agonists on the proximal stomach, but the pathway involved and the symptomatic consequences have been incompletely elucidated. AIMS: To study whether motilin enhances proximal stomach tone and enhances meal-induced satiety and to evaluate whether this effect involves a cholinergic pathway. METHODS: A gastric barostat was used to study, in healthy subjects, the effect of motilin (300 ng/kg/30 min i.v.) or saline on fasting gastric fundus tone and on post-prandial relaxation. To evaluate the involvement of a cholinergic pathway, atropine (12 microg/kg/h) was administered intravenously simultaneously with or before and during motilin infusion in the fasting state. Finally, a satiety drinking test was performed in 21 subjects twice after pretreatment with placebo or motilin and with placebo or atropine. RESULTS: Administration of motilin caused a significant increase of fasting fundus tone expressed as decrease of the mean balloon volume (324 +/- 60 mL vs 213 +/- 62 mL, p < 0.05). Simultaneous administration of atropine and motilin did not generate a significant volume change (192 +/- 60 mL vs 181 +/- 83 mL, NS), but pretreatment with atropine alone induced a relaxation, and when motilin was added this revealed an ongoing contraction (192 +/- 24 mL vs 136 +/- 21 mL, p < or = 0.05). Motilin infusion also inhibited gastric accommodation (p < or = 0.05 vs placebo) and increased satiety during a satiety drinking test (p < or = 0.05 vs placebo). CONCLUSIONS: Administration of motilin causes a contraction of the proximal stomach in humans and increases meal-induced satiety. The effect of motilin is atropine-resistant and involves a direct muscular pathway or a non-cholinergic neural pathway.

Altered gastrointestinal and metabolic function in the GPR39-obestatin receptor-knockout mouse.

BACKGROUND & AIMS: The G-protein-coupled receptor GPR39 is a member of a family that includes the receptors for ghrelin and motilin. Recently the peptide obestatin was identified as a natural ligand for GPR39. The objective of this study was to gain insight into the biological function of the GPR39 receptor. METHODS: GPR39(-/-) mice were generated and analyzed. RESULTS: Endogenous GPR39 expression was detected in the brain (septum-amygdala) and the gastrointestinal system (parietal cells, enterocytes, neurons, and pancreas). Gastric emptying of a solid meal (measured by the (14)C octanoic breath test) in GPR39(-/-) mice was accelerated significantly with a gastric half-emptying time of 49.5 +/- 2.2 minutes compared with 86.9 +/- 8.4 minutes in GPR39(+/+) mice. A more effective expulsion of distally located pellets (30%-75% of length) was observed in the colon of GPR39(-/-) mice. Four hours after pylorus ligation, the volume of gastric secretion was increased significantly (GPR39(-/-): 638 +/- 336 microL; GPR39(+/+): 225 +/- 170 microL), but gastric acid secretion was unchanged. The mature body weight and body fat composition of GPR39(-/-) mice was significantly higher compared with GPR39(+/+) mice, but this was not related to hyperphagia because 24-hour food intake did not differ between both genotypes. In contrast, deficiency of the GPR39 receptor led to reduced hyperphagia after fasting. The cholesterol levels were increased significantly in the GPR39(-/-) mice. CONCLUSIONS: Our data partially confirm and extend the described in vivo effects of obestatin and suggest that this peptide plays a functional role in the regulation of gastrointestinal and metabolic function through interaction with the GPR39 receptor.

Serotonergic modulation of murine fundic tone.

Fundic tone is maintained through a balance of excitatory and inhibitory input to fundic smooth muscle. The aim of this study was to determine the role of serotonin (5-HT) and 5-HT receptors in modulating murine fundic tone. Muscle strips were prepared from the murine fundus. Intracellular recordings were made from circular smooth muscle cells, and the effects of 5-HT on tone and excitatory and inhibitory junction potentials evoked by electrical field stimulation (EFS) were determined. 5-HT induced a concentration-dependent contraction and smooth muscle depolarization that was tetrodotoxin resistant. The 5-HT(1B/D) receptor antagonists GR-127935 and BRL-155172 significantly inhibited 5-HT-induced contractions. The 5-HT(1B/D) agonist sumatriptan contracted murine fundic muscle. The 5-HT(1A) receptor agonist buspirone relaxed fundic smooth muscle, and the relaxation was inhibited by WAY-100135 but not by N(omega)-nitro-l-arginine or tetrodotoxin. 5-HT enhanced both the excitatory and inhibitory responses to EFS. The 5-HT(3) receptor antagonist MDL-72222 partly inhibited both the excitatory and inhibitory response elicited by EFS, whereas the 5-HT(4) receptor antagonist GR-113808 partly inhibited the EFS-evoked inhibitory response. The 5-HT reuptake inhibitor fluoxetine contracted smooth muscle strips, a contraction that was partially inhibited by GR-127935 and abolished by tetrodotoxin. In conclusion, the data suggest that 5-HT modulates murine fundic contractile activity through several different receptor subtypes. Sustained release of 5-HT maintains fundic tone through postjunctional 5-HT(1B/D) receptors. 5-HT(3) receptors modulate excitatory neural input to murine fundic smooth muscle, and both 5-HT(3) and 5-HT(4) receptors modulate inhibitory neural input to murine fundic smooth muscle.

Effect of modulation of serotonergic, cholinergic, and nitrergic pathways on murine fundic size and compliance measured by ultrasonomicrometry.

Reduced fasting or postprandial gastric volumes have been implicated in the pathophysiology of functional dyspepsia. The mechanisms that underlie the control of gastric fundic volume are incompletely understood, partly because of an inability to accurately measure fundic volume in vivo in small animals. Small animals are useful models to evaluate mechanisms, e.g., in knockout animals. The aim of this study was to determine whether an ultrasonometric technique accurately monitors fundic contraction and relaxation in mice in vivo and to determine the effect of modulation of cholinergic, nitrergic, and serotonergic pathways on fundic size and compliance in the intact mouse innervated stomach. Two to four piezoelectric crystals (diameter 1 mm, 24-microm resolution) were glued to the serosal side of fundus and used to measure distance. Validation studies showed excellent correlation between measured changes and actual changes in distances between crystals and excellent reproducibility. The expected responses to pharmacological modulation with bethanechol and nitroglycerin were demonstrated. Atropine increased the distance between the crystals, suggesting a baseline cholinergic regulation of fundic volume. Bethanechol, Nomega-nitro-L-arginine, and the 5-HT1B/D agonist sumatriptan decreased the distance between the crystals, suggesting fundic contraction. Atropine, nitroglycerin, and buspirone caused an increase in intercrystal distance consistent with fundic relaxation. Fundic compliance was investigated by changing intragastric pressure via an implanted catheter. Sumatriptan increased compliance, whereas buspirone increased the distance between crystals but did not change compliance. The data suggest that ultrasonomicrometry is a useful tool that can reproducibly and accurately measure changes in fundic size and the response to pharmacological agents.

Dissecting the role of sodium currents in visceral sensory neurons in a model of chronic hyperexcitability using Nav1.8 and Nav1.9 null mice.

Tetrodotoxin-resistant (TTX-R) sodium currents have been proposed to underlie sensory neuronal hyperexcitability in acute inflammatory models, but their role in chronic models is unknown. Since no pharmacological tools to separate TTX-R currents are available, this study employs Na(v)1.8 and Na(v)1.9 null mice to evaluate these currents roles in a chronic hyperexcitability model after the resolution of an inflammatory insult. Transient jejunitis was induced by infection with Nippostrongylus brasiliensis (Nb) in Na(v)1.9 and Na(v)1.8 null, wild-type and naïve mice. Retrogradely labelled dorsal root ganglia (DRG) neurons were harvested on day 20-24 post-infection for patch clamp recording. Rheobase and action potential (AP) parameters were recorded as measures of excitability, and Na(v)1.9 and Na(v)1.8 currents were recorded. DRG neuronal excitability was significantly increased in post-infected mice compared to sham animals, despite the absence of ongoing inflammation (sham = 1.9 +/- 0.3, infected = 3.6 +/- 0.7 APs at 2x rheobase, P = 0.02). Hyperexcitability was associated with a significantly increased amplitude of TTX-R currents. Hyperexcitability was maintained in Na(v)1.9(-/-) mice, but hyperexcitability was absent and APs were blunted in Na(v)1.8(-/-) mice. This study identifies a critical role for Na(v)1.8 in chronic post-infectious visceral hyperexcitability, with no contribution from Na(v)1.9. Nb infection-induced hyperexcitability is not observed in Na(v)1.8(-/-) mice, but is still present in Na(v)1.9(-/-) mice. It is not clear whether hyperexcitability is due to a change in the function of Na(v)1.8 channels or a change in the number of Na(v)1.8 channels.

Demonstration of a specific site of covalent labeling of the human motilin receptor using a biologically active photolabile motilin analog.

The motilin receptor belongs to a group of class I G protein-coupled receptors that also includes the growth hormone secretagogue and ghrelin receptors. These represent clinically useful targets for pharmacotherapy. Their potentially unique structures and the molecular basis of their binding are not yet clear. We previously reported the initial affinity labeling of a region within this receptor (a cyanogen bromide fragment extending from the first to the second extracellular loop) using a position 1 photolabile motilin analog. To extend our understanding of the molecular basis of motilin binding, we have developed an additional radioiodinatable motilin analog probe having site of covalent attachment in position 5. This was a full agonist that bound to the motilin receptor specifically and with high affinity, and that efficiently established a single covalent bond to its receptor. Sequential chemical and enzymatic cleavage of labeled wild-type and mutant motilin receptor constructs established that the region of labeling was within the third extracellular loop. This was further localized to Phe(332) using radiochemical Edman degradation sequencing. These data provide the first spatial approximation constraint that can be used in the docking of this peptide ligand to its receptor. We hope that a series of such constraints can be determined to provide adequate structural information to begin to elucidate the conformation of this agonist-bound receptor and to ultimately be useful in the rational design of drugs acting at this important target.

Prevalence and socioeconomic impact of upper gastrointestinal disorders in the United States: results of the US Upper Gastrointestinal Study.

BACKGROUND & AIMS: This study examined the prevalence of upper gastrointestinal (GI) symptoms and symptom groupings and determined impact on disability days in a nationally representative US sample. METHODS: A telephone survey of 21,128 adults was conducted including questions about the presence of upper GI symptoms during the past 3 months. Respondents were categorized as symptomatic (ie, reported GI symptoms once per month) or asymptomatic. The survey included questions about missed work, leisure activity, or household activity days. Symptom groupings were identified by using factor analysis, and cluster analysis was used to assign respondents into distinct groups on the basis of these symptom groupings. RESULTS: The prevalence of an average of 1 or more upper GI symptoms during the past 3 months was 44.9%. The most common symptoms experienced during the past 3 months were early satiety, heartburn, and postprandial fullness. Factor analysis identified 4 symptom groupings: (1) heartburn/regurgitation; (2) nausea/vomiting; (3) bloating/abdominal pain; and (4) early satiety/loss of appetite. Five respondent clusters were identified; the largest clusters were primarily early satiety/fullness (44%) and gastroesophageal reflux disease-like symptoms (28%). Two small clusters reflected nausea and vomiting (7%) and a heterogeneous symptom profile (4%). Symptomatic respondents reported significantly more missed work, leisure, and household activity days than asymptomatic respondents (all P < .0001). CONCLUSIONS: Factor analysis separated GI symptoms into groupings reflecting gastroesophageal reflux disease and dyspepsia: early satiety, postprandial fullness, and loss of appetite; bloating and abdominal pain/discomfort; and nausea and vomiting. These upper GI symptoms were associated with significant loss of work and activity days.