Elice M. Brooks

Activation of colonic mucosal 5-HT(4) receptors accelerates propulsive motility and inhibits visceral hypersensitivity.

BACKGROUND & AIMS 5-hydroxytryptamine receptor (5-HT(4)R) agonists promote gastrointestinal motility and attenuate visceral pain, but concerns about adverse reactions have restricted their availability. We tested the hypotheses that 5-HT(4) receptors are expressed in the colonic epithelium and that 5-HT(4)R agonists can act intraluminally to increase motility and reduce visceral hypersensitivity. METHODS Mucosal expression of the 5-HT(4)R was evaluated by reverse-transcriptase polymerase chain reaction and immunohistochemical analysis of tissues from 5-HT(4)R(BAC)-enhanced green fluorescent protein mice. Amperometry, histology, and short-circuit current measurements were used to study 5-HT, mucus, and Cl(-) secretion, respectively. Propulsive motility was measured in guinea pig distal colon, and visceromotor responses were recorded in a rat model of colonic hypersensitivity. 5-HT(4)R compounds included cisapride, tegaserod, naronapride, SB204070, and GR113808. RESULTS Mucosal 5-HT(4) receptors were present in the small and large intestines. In the distal colon, 5-HT(4) receptors were expressed by most epithelial cells, including enterochromaffin and goblet cells. Stimulation of 5-HT(4)Rs evoked mucosal 5-HT release, goblet cell degranulation, and Cl(-) secretion. Luminal administration of 5-HT(4)R agonists accelerated propulsive motility; a 5-HT(4)R antagonist blocked this effect. Bath application of 5-HT(4)R agonists did not affect motility. Oral or intracolonic administration of 5-HT(4)R agonists attenuated visceral hypersensitivity. Intracolonic administration was more potent than oral administration, and was inhibited by a 5-HT(4)R antagonist. CONCLUSIONS Mucosal 5-HT(4) receptor activation can mediate the prokinetic and antinociceptive actions of 5-HT(4)R agonists. Colon-targeted, intraluminal delivery of 5-HT(4)R agonists might be used to promote motility and alleviate visceral pain, while restricting systemic bioavailability and resulting adverse side effects.

Serotonin signaling is altered in irritable bowel syndrome with diarrhea but not in functional dyspepsia in pediatric age patients.

BACKGROUND & AIMS In adults, irritable bowel syndrome (IBS) and functional dyspepsia (FD) are chronic conditions that often start during childhood. We investigated mucosal serotonin (5-HT) signaling in children with the idea that data from subjects with a shorter history may improve our understanding of underlying pathophysiological mechanisms. METHODS Ninety-eight children undergoing gastroscopy or colonoscopy were studied prospectively. Biopsy specimens were evaluated for inflammation, enterochromaffin cell numbers, 5-HT content, and messenger RNA (mRNA) levels for the synthetic enzyme, tryptophan hydroxylase 1, and the serotonin transporter (SERT) were assessed by quantitative real-time reverse-transcription polymerase chain reaction. RESULTS Data from 12 children with IBS and 17 with FD were compared with age-matched controls (12 with rectal biopsies and 12 with gastric biopsies) and with subjects with organic disorders. In patients with FD, a small number of immune cells were observed in the gastric mucosa in half of the patients, but no abnormalities with respect to the 5-HT pathway were identified. In patients with IBS, no differences were detected between patients and controls regarding intraepithelial lymphocytes and CD3+ cells in the lamina propria although all patients showed at least a slight inflammatory infiltrate. In the IBS samples, higher 5-HT content (P < .01) and lower SERT mRNA (P < .05) were detected as compared with controls. Severe inflammation in the colonic mucosa had a high impact on 5-HT signaling with a significant decrease in enterochromaffin cells (P < .01) and 5-HT content (P < .01) and a high SERT mRNA expression (P < .01). CONCLUSIONS These results confirm the role of 5-HT signaling in IBS in children and argue against such a role in FD.

Comparative Decellularization and Recellularization of Normal versus Emphysematous Human Lungs

Acellular whole human lung scaffolds represent a unique opportunity for ex vivo tissue engineering. However, it remains unclear whether lungs from individuals with chronic lung diseases such as chronic obstructive pulmonary disease (COPD) can be appropriately decellularized and recellularized. To assess this, cadaveric human lungs from normal (non-smoking) patients and from patients with COPD (smoking history) were decellularized and found by histochemical and immunohistochemical staining, electron microscopy, and mass spectrometry to retain characteristic histological architecture and extracellular matrix components (ECM) reflecting either normal or COPD, particularly emphysematous, origin. Inoculation of human bronchial epithelial cells, endothelial progenitor cells, bone marrow-derived mesenchymal stem cells, and lung fibroblasts via airway or vascular routes into small, excised segments of the decellularized lungs demonstrated that normal lung scaffolds robustly supported initial engraftment and growth of each cell type for up to one month. In contrast, despite initial binding, all cell types inoculated into decellularized emphysematous lungs did not survive beyond one week. However, cell attachment and proliferation on solubilized ECM homogenates of decellularized normal and emphysematous lungs coated onto tissue culture plates was comparable and not impaired, suggesting that the 3-dimensional decellularized emphysematous scaffolds may lack the necessary ECM architecture to support sustained cell growth.

Three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration

Acellular scaffolds from complex whole organs such as lung are being increasingly studied for ex vivo organ generation and for in vitro studies of cell-extracellular matrix interactions. We have established effective methods for efficient de and recellularization of large animal and human lungs including techniques which allow multiple small segments (∼ 1-3 cm(3)) to be excised that retain 3-dimensional lung structure. Coupled with the use of a synthetic pleural coating, cells can be selectively physiologically inoculated via preserved vascular and airway conduits. Inoculated segments can be further sliced for high throughput studies. Further, we demonstrate thermography as a powerful noninvasive technique for monitoring perfusion decellularization and for evaluating preservation of vascular and airway networks following human and porcine lung decellularization. Collectively, these techniques are a significant step forward as they allow high throughput in vitro studies from a single lung or lobe in a more biologically relevant, three-dimensional acellular scaffold.

Changes in colonic motility and the electrophysiological properties of myenteric neurons persist following recovery from trinitrobenzene sulfonic acid colitis in the guinea pig

Abstract  Persistent changes in gastrointestinal motility frequently accompany the resolution of colitis, through mechanisms that remain to be determined. Trinitrobenzene sulfonic acid (TNBS) colitis in the guinea pig decreases the rate of propulsive motility, causes hyperexcitability of AH neurons, and induces synaptic facilitation. The changes in motility and AH neurons are sensitive to cyclooxygenase‐2 (COX‐2) inhibition. The aim of this investigation was to determine if the motility and neurophysiological changes persist following recovery from colitis. Evaluations of inflammation, colonic motility and intracellular electrophysiology of myenteric neurons 8 weeks after TNBS administration were performed and compared to matched control conditions. Myeloperoxidase levels in the colons were comparable to control levels 56 days after TNBS treatment. At this time point, the rate of colonic motility was decreased relative to controls following treatment with TNBS alone or TNBS plus a COX‐2 inhibitor. Furthermore, the electrical properties of AH neurons and fast synaptic potentials in S neurons were significantly different from controls and comparable to those detected during active inflammation. Collectively, these data suggest that altered myenteric neurophysiology initiated during active colitis persists long term, and provide a potential mechanism underlying altered gut function in individuals during remission from inflammatory bowel disease.

Mesenchymal Stromal Cells Mediate Aspergillus Hyphal Extract-Induced Allergic Airway Inflammation by Inhibition of the Th17 Signaling Pathway

Systemic administration of mesenchymal stromal cells (MSCs) suppresses airway inflammation and methacholine‐induced airway hyper‐responsiveness (AHR) in mouse models of T helper cell (Th) type 2‐mediated eosinophilic allergic airway inflammation (AAI); however, the efficacy of MSCs in mouse models of severe Th17‐mediated neutrophilic AAI has not yet been demonstrated. We assessed MSC effects in a mouse model of mixed Th2/Th17 AAI produced by mucosal exposure to Aspergillus fumigatus hyphal extract (AHE). Following sensitization produced by oropharyngeal AHE administration, systemic (tail vein) administration of syngeneic MSCs on the first day of challenge significantly reduced acute AHR predominantly through reduction of Th17‐mediated airway inflammation. In parallel experiments, MSCs also mitigated AHR when administered during recurrent challenge 10 weeks after initial sensitization and challenge through reduction in systemic Th17‐mediated inflammation. Investigation into potential mechanistic actions of MSCs in this model demonstrated that although T regulatory cells were increased in all AHE‐treated mice, MSC administration did not alter T regulatory cell numbers in either the acute or recurrent model. Differential induction of interleukin‐17a secretion was observed in ex vivo restimulation of mediastinal lymph node mixed‐cell cytokine analyses. Although the mechanisms by which MSCs act to decrease inflammation and AHR in this model are not yet fully elucidated, decrease in Th17‐mediated airway inflammation appears to play a significant role. These results provide a basis for further investigations of MSC administration as a potential therapeutic approach for severe refractory neutrophilic asthma.

Molecular description of three macro-deletions and an Alu-Alu recombination-mediated duplication in the HPRT gene in four patients with Lesch-Nyhan disease

Mutations in the HPRT gene cause a spectrum of diseases that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. The extreme phenotype is termed Lesch-Nyhan syndrome. In 271 cases in which the germinal HPRT mutation has been characterized, 218 different mutations have been found. Of these, 34 (13%) are large- (macro-) deletions of one exon or greater and four (2%) are partial gene duplications. The deletion breakpoint junctions have been defined for only three of the 34 macro-deletions. The molecular basis of two of the four duplications has been defined. We report here the breakpoint junctions for three new deletion mutations, encompassing exons 4-8 (20033bp), exons 4 and 5 (13307bp) and exons 5 and 6 (9454bp), respectively. The deletion breakpoints were defined by a combination of long polymerase chain reaction (PCR) amplifications, and conventional PCR and DNA sequencing. All three deletions are the result of non-homologous recombinations. A fourth mutation, a duplication of exons 2 and 3, is the result of an Alu-mediated homologous recombination between identical 19bp sequences in introns 3 and 1. In toto, two of three germinal HPRT duplication mutations appear to have been caused by Alu-mediated homologous recombination, while only one of six deletion mutations appears to have resulted from this type of recombination mechanism. The other five deletion mutations resulted from non-homologous recombination. With this admittedly limited number of characterized macro-mutations, Alu-mediated unequal homologous recombinations account for at least 8% (3 of 38) of the macro-alterations and 1% (3 of 271) of the total HPRT germinal mutations.

Mucosal Serotonin Signaling Is Altered in Chronic Constipation but Not in Opiate-Induced Constipation

OBJECTIVES:Changes in mucosal serotonin (5-HT) signaling have been detected in a number of functional and inflammatory disorders of the gastrointestinal (GI) tract. This study was undertaken to determine whether chronic constipation (CC) is associated with disordered 5-HT signaling and to evaluate whether constipation caused by opiate use causes such changes.METHODS:Human rectal biopsy samples were obtained from healthy volunteers, individuals with idiopathic CC, and individuals taking opiate medication with or without occurrence of constipation. EC cells were identified by 5-HT immunohistochemistry. 5-HT content and release levels were determined by enzyme immunoassay, and mRNA levels for the synthetic enzyme tryptophan hydroxylase-1 (TpH-1) and serotonin-selective reuptake transporter (SERT) were assessed by quantitative real-time reverse transcription PCR.RESULTS:CC was associated with increases in TpH-1 transcript, 5-HT content, and 5-HT release under basal and stimulated conditions, whereas EC cell numbers and SERT transcript levels were not altered. No changes in these elements of 5-HT signaling were detected in opiate-induced constipation (OIC).CONCLUSIONS:These findings demonstrate that CC is associated with a pattern of altered 5-HT signaling that leads to increased 5-HT availability but does not involve a decrease in SERT expression. It is possible that increased 5-HT availability due to increased synthesis and release contributes to constipation due to receptor desensitization. Furthermore, the finding that elements of 5-HT signaling were not altered in the mucosa of individuals with OIC indicates that constipation as a condition does not lead to compensatory changes in 5-HT synthesis, release, or signal termination.

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Novel promoter and alternate transcription start site of the human serotonin reuptake transporter in intestinal mucosa.

Abstract  Selective serotonin‐reuptake inhibitors are therapies for psychological and bowel disorders, but produce adverse effects in the non‐targeted system. To determine whether human serotonin‐selective reuptake transporter (SERT) transcripts in the intestine are different from the brain, rapid amplification of cDNA ends, primer extension and RT‐PCR assays were used to evaluate SERT transcripts from each region. Potential SLC6A4 gene promoter constructs were evaluated with a secreted alkaline phosphatase reporter assay. A novel transcript of the human SLC6A4 gene was discovered that predominates in the intestine, and differs from previous transcripts in the 5′‐untranslated region. The distinct transcriptional start site and alternate promoter suggest that gastrointestinal SERT can be differentially regulated from brain SERT, may explain why the polymorphism in the previously identified promoter is associated with affective disorders, but not associated with gastrointestinal dysfunction, and suggest the intriguing possibility of the development of site‐specific therapeutics for SERT regulation in the treatment of multiple disorders.