Prebiotics counteract the morphological and functional changes secondary to chronic cisplatin exposition in the proximal colon of mice.

Cisplatin is an antimitotic drug able to cause acute and chronic gastrointestinal side effects. Acute side effects are attributable to mucositis while chronic ones are due to neuropathy. Cisplatin has also antibiotic properties inducing dysbiosis which enhances the inflammatory response, worsening local damage. Thus, a treatment aimed at protecting the microbiota could prevent or reduce the toxicity of chemotherapy. Furthermore, since a healthy microbiota enhances the effects of some chemotherapeutic drugs, prebiotics could also improve this drug effectiveness. We investigated whether chronic cisplatin administration determined morphological and functional alterations in mouse proximal colon and whether a diet enriched in prebiotics had protective effects. The results showed that cisplatin caused lack of weight gain, increase in kaolin intake, decrease in stool production and mucus secretion. Prebiotics prevented increases in kaolin intake, changes in stool production and mucus secretion, but had no effect on the lack of weight gain. Moreover, cisplatin determined a reduction in amplitude of spontaneous muscular contractions and of Connexin (Cx)43 expression in the interstitial cells of Cajal, changes that were partially prevented by prebiotics. In conclusion, the present study shows that daily administration of prebiotics, likely protecting the microbiota, prevents most of the colonic cisplatin-induced alterations.

Glucagon-like Peptide-2 Depresses Ileal Contractility in Preparations from Mice through Opposite Modulatory Effects on Nitrergic and Cholinergic Neurotransmission.

Glucagon-like peptide-2 (GLP-2) has been reported to influence gastrointestinal motor responses, exerting a modulatory role on enteric neurotransmission. To our knowledge, no data on GLP-2 effects on the motility of the isolated ileum are available; therefore, we investigated whether GLP-2 affects the contractile activity of mouse ileal preparations and the neurotransmitters engaged. Ileal preparations showed tetrodotoxin (TTX)- and atropine-insensitive spontaneous contractile activity, which was unaffected by the nitric oxide synthesis inhibitor, L-NNA. GLP-2 depressed the spontaneous contractility, an effect that was abolished by TTX or L-NNA and not influenced by atropine. Electrical field stimulation induced TTX- and atropine-sensitive contractile responses, which were reduced in amplitude by GLP-2 even in the presence of L-NNA. Immunohistochemical results showed a significant increase in nNOS-positive fibers in the ileal muscle wall and a significant decrease in ChAT-positive myenteric neurons in GLP-2-exposed preparations. The present results offer the first evidence that GLP-2 acts on ileal preparations. The hormone appears to depress ileal contractility through a dual opposite modulatory effect on inhibitory nitrergic and excitatory cholinergic neurotransmission. From a physiological point of view, it could be hypothesized that GLP-2 inhibitory actions on ileal contractility can increase transit time, facilitating nutrient absorption.

Development and Evaluation of Khellin-loaded Microemulgel for Dermatological Applications.

Microemulsions are optically nanosized emulsions, isotropic and thermodynamically stable. They represent versatile drug delivery systems with high potential because they can be administered regardless of route. In the present study, we report on the formulation of a microemulsion made with glycerol (2.25%), Labrasol (20.25%) vitamin E acetate (2.50%), and water (75.00%), which was developed using the pseudo-ternary phase diagram. Globules of the microemulsion had PdI less than 0.25 and size of about 17 nm, evaluated by DLS analysis. These values did not change after loading khellin, a natural lipophilic molecule with interesting biological activities, used as a model of lipophilic drug. Carboxymethyl cellulose was selected as gelling polymer to obtain a microemulgel. Viscosity was 22 100.0 ± 1555.6 mPas·s at 21 ± 2 °C, while it was 8916.5 ± 118.1 mPas·s at 35 ± 2 °C, remaining stable over time. Khellin recovery was 93.16 ± 4.39% and was unchanged after 4 weeks of storage (93.23 ± 2.14%). The pH was 6.59 ± 0.19 and it was found to be 6.42 ± 0.34 at the end of the storage lifetime. The diffusion of khellin from the developed formulation was prolonged over an extended period. Based on overall results and due to the dermatological properties of the ingredients of the formulation, the developed microemulgel loaded with khellin is very promising and suitable for skin care applications.

Otilonium Bromide Prevents Cholinergic Changes in the Distal Colon Induced by Chronic Water Avoidance Stress, a Rat Model of Irritable Bowel Syndrome.

Irritable Bowel syndrome (IBS) is a highly widespread gastrointestinal disorder whose symptomatology mainly affect the large intestine. Among the risk factors, psychosocial stress is the most acknowledged. The repeated water avoidance stress (rWAS) is considered an animal model of psychosocial stress that is capable of mimicking IBS. Otilonium bromide (OB), which is orally administered, concentrates in the large bowel and controls most of the IBS symptoms in humans. Several reports have shown that OB has multiple mechanisms of action and cellular targets. We investigated whether the application of rWAS to rats induced morphological and functional alterations of the cholinergic neurotransmission in the distal colon and whether OB prevented them. The results demonstrated that rWAS affects cholinergic neurotransmission by causing an increase in acid mucin secretion, in the amplitude of electrically evoked contractile responses, abolished by atropine, and in the number of myenteric neurons expressing choline acetyltransferase. OB counteracted these changes and also showed an intrinsic antimuscarinic effect on the post-synaptic muscular receptors. We assume that the rWAS consequences on the cholinergic system are linked to corticotrophin-releasing factor-1 (CRF1) receptor activation by the CRF hypothalamic hormone. OB, by interfering with the CFR/CRFr activation, interrupted the cascade events responsible for the changes affecting the rWAS rat colon.

Telocytes and Macrophages in the Gut: From Morphology to Function, Do the Two Cell Types Interact with Each Other? Which Helps Which?

Telocytes and macrophages are ubiquitous cells located in loose connective tissues and share the same mesenchymal origin. Despite these common elements, depending on where they reside, these two cell types are profoundly different in terms of their morphology and functions. The purpose of this review is to provide an update on the knowledge regarding telocytes and macrophages in the gut, where their presence and significance have long been underestimated or misunderstood. The focus will be on the possibility that these two cell types interact with each other and on the potential meaning of these interactions. Based on the complexity of the topic, the variety of possible methodological approaches and the expertise of the author, the point of view in the discussion of the literature data will be mainly morphological. Furthermore, considering the relatively recent period in which these cell types have acquired a primary role in gastrointestinal functions, the attention will be greatly confined to those articles published in the last decade. The microbiota, another main protagonist in this context, will be mentioned only in passing. It is hoped that this review, although not exhaustive, will highlight the importance of macrophages and telocytes in the complex mechanisms that ensure intestinal functions.

Otilonium Bromide treatment prevents nitrergic functional and morphological changes caused by chronic stress in the distal colon of a rat IBS model.

Irritable bowel syndrome (IBS) is a highly prevalent gastrointestinal disorder characterized by periods of remission and exacerbation. Among the risk factors to develop IBS, psychosocial stress is widely acknowledged. The water avoidance stress repeatedly applied (rWAS) is considered effective to study IBS etio-pathogenesis. Otilonium bromide (OB), a drug with multiple mechanisms of action, is largely used to treat IBS patients. Orally administered, it concentrates in the large bowel and significantly ameliorates the IBS symptomatology. Presently, we tested whether rWAS rats developed neuro-muscular abnormalities in the distal colon and whether OB treatment prevented them. The investigation was focussed on the nitrergic neurotransmission by combining functional and morphological methodologies. The results confirm rWAS as reliable animal model to investigate the cellular mechanisms responsible for IBS: exposure to one-hour psychosocial stress for 10 days depressed muscle contractility and increased iNOS expression in myenteric neurons. OB treatment counteracted these effects. We hypothesize that these effects are due to the corticotropin-releasing factor (CRF) release, the main mediator of the psychosocial stress, followed by a CRF1receptor activation. OB, that was shown to prevent CRF1r activation, reasonably interrupted the cascade events that bring to the mechanical and immunohistochemical changes affecting rWAS rat colon.

Nitric Oxide: From Gastric Motility to Gastric Dysmotility.

It is known that nitric oxide (NO) plays a key physiological role in the control of gastrointestinal (GI) motor phenomena. In this respect, NO is considered as the main non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitter responsible for smooth muscle relaxation. Moreover, many substances (including hormones) have been reported to modulate NO production leading to changes in motor responses, further underlying the importance of this molecule in the control of GI motility. An impaired NO production/release has indeed been reported to be implicated in some GI dysmotility. In this article we wanted to focus on the influence of NO on gastric motility by summarizing knowledge regarding its role in both physiological and pathological conditions. The main role of NO on regulating gastric smooth muscle motor responses, with particular reference to NO synthases expression and signaling pathways, is discussed. A deeper knowledge of nitrergic mechanisms is important for a better understanding of their involvement in gastric pathophysiological conditions of hypo- or hyper-motility states and for future therapeutic approaches. A possible role of substances which, by interfering with NO production, could prove useful in managing such motor disorders has been advanced.

The Telocytes: Ten Years after Their Introduction in the Scientific Literature. An Update on Their Morphology, Distribution, and Potential Roles in the Gut.

Ten years ago, the term 'telocyte' was introduced in the scientific literature to describe a 'new' cell type described in the connective tissue of several organs by Popescu and Faussone-Pellegrini (2010). Since then, 368 papers containing the term 'telocyte' have been published, 261 of them in the last five years. These numbers underscore the growing interest in this cell type in the scientific community and the general acceptance of the name telocyte to indicate this interstitial cell. Most of these studies, while confirming the importance of transmission electron microscopy to identify the telocytes with certainty, highlight the variability of their immune phenotypes. This variability was interpreted as due to (i) the ability of the telocytes to adapt to the different sites in which they reside; (ii) the distinct functions they are likely to perform; and (iii) the existence of telocyte subtypes. In the present paper, an overview of the last 10 years of literature on telocytes located in the gut will be attempted, confining the revision to the morphological findings. A distinct chapter will be dedicated to the recently hypothesized role of the telocytes the intestinal mucosa. Through this review, it will be shown that telocytes, despite their variability, are a unique interstitial cell.

GLP-2 Prevents Neuronal and Glial Changes in the Distal Colon of Mice Chronically Treated with Cisplatin.

Cisplatin is a chemotherapeutic agent widely used for the treatment of solid cancers. Its administration is commonly associated with acute and chronic gastrointestinal dysfunctions, likely related to mucosal and enteric nervous system (ENS) injuries, respectively. Glucagon-like peptide-2 (GLP-2) is a pleiotropic hormone exerting trophic/reparative activities on the intestine, via antiapoptotic and pro-proliferating pathways, to guarantee mucosal integrity, energy absorption and motility. Further, it possesses anti-inflammatory properties. Presently, cisplatin acute and chronic damages and GLP-2 protective effects were investigated in the mouse distal colon using histological, immunohistochemical and biochemical techniques. The mice received cisplatin and the degradation-resistant GLP-2 analog ([Gly2]GLP-2) for 4 weeks. Cisplatin-treated mice showed mucosal damage, inflammation, IL-1ß and IL-10 increase; decreased number of total neurons, ChAT- and nNOS-immunoreactive (IR) neurons; loss of SOX-10-IR cells and reduced expression of GFAP- and S100ß-glial markers in the myenteric plexus. [Gly2]GLP-2 co-treatment partially prevented mucosal damage and counteracted the increase in cytokines and the loss of nNOS-IR and SOX-10-IR cells but not that of ChAT-IR neurons. Our data demonstrate that cisplatin causes mucosal injuries, neuropathy and gliopathy and that [Gly2]GLP-2 prevents these injuries, partially reducing mucosal inflammation and inducing ENS remodeling. Hence, this analog could represent an effective strategy to overcome colonic injures induced by cisplatin.

Nerve sprouting and neurogenic inflammation characterize the neurogenic detrusor overactive bladder of patients no longer responsive to drug therapies.

Urothelium and Lamina Propria (LP) are considered an integrate sensory system which is able to control the detrusor activity. Complete supra-sacral spinal cord lesions cause Neurogenic Detrusor Overactivity (NDO) whose main symptoms are urgency and incontinence. NDO therapy at first consists in anti-muscarinic drugs; secondly, in intra-vesical injection of botulinum toxin. However, with time, all the patients become insensitive to the drugs and decide for cystoplastic surgery. With the aim to get deeper in both NDO and drug's efficacy lack pathogenesis, we investigated the innervation, muscular and connective changes in NDO bladders after surgery by using morphological and quantitative methodologies. Bladder innervation showed a significant global loss associated with an increase in the nerve endings located in the upper LP where a neurogenic inflammation was also present. Smooth muscle cells (SMC) anomalies and fibrosis were found in the detrusor. The increased innervation in the ULP is suggestive for a sprouting and could condition NDO evolution and drug efficacy length. Denervation might cause the SMC anomalies responsible for the detrusor altered contractile activity and intra-cellular traffic and favour the appearance of fibrosis. Inflammation might accelerate these damages. From the clinical point of view, an early anti-inflammatory treatment could positively influence the disease fate.

Adaptive changes of telocytes in the urinary bladder of patients affected by neurogenic detrusor overactivity.

Urinary bladder activity involves central and autonomic nervous systems and bladder wall. Studies on the pathogenesis of voiding disorders such as the neurogenic detrusor overactivity (NDO) due to suprasacral spinal cord lesions have emphasized the importance of an abnormal handling of the afferent signals from urothelium and lamina propria (LP). In the LP (and detrusor), three types of telocytes (TC) are present and form a 3D-network. TC are stromal cells able to form the scaffold that contains and organizes the connective components, to serve as guide for tissue (re)-modelling, to produce trophic and/or regulatory molecules, to share privileged contacts with the immune cells. Specimens of full thickness bladder wall from NDO patients were collected with the aim to investigate possible changes of the three TC types using histology, immunohistochemistry and transmission electron microscopy. The results show that NDO causes several morphological TC changes without cell loss or network interruption. With the exception of those underlying the urothelium, all the TC display signs of activation (increase in Caveolin1 and caveolae, αSMA and thin filaments, Calreticulin and amount of cisternae of the rough endoplasmic reticulum, CD34, euchromatic nuclei and large nucleoli). In all the specimens, a cell infiltrate, mainly consisting in plasma cells located in the vicinity or taking contacts with the TC, is present. In conclusion, our findings show that NDO causes significant changes of all the TC. Notably, these changes can be interpreted as TC adaptability to the pathological condition likely preserving each of their peculiar functions.

Repeated otilonium bromide administration prevents neurotransmitter changes in colon of rats underwent to wrap restraint stress.

Otilonium bromide (OB) is a spasmolytic drug successfully used for the treatment of irritable bowel syndrome (IBS). Its efficacy has been attributed to the block of L- and T-type Ca2+ channels and muscarinic and tachykinin receptors in the smooth muscle. Furthermore, in healthy rats, repeated OB administration modified neurotransmitter expression and function suggesting other mechanisms of action. On this basis, we investigated whether repeated OB treatment prevented the functional and neurochemical changes observed in the colon of rats underwent to wrap restrain stress (WRS) a psychosocial stressor considered suitable to reproduce the main IBS signs and symptoms. In control, WRS and OB/WRS rats functional parameters were measured in vivo and morphological investigations were done ex vivo in the colon. The results showed that OB counteracts most of the neurotransmitters changes caused by WRS. In particular, the drug prevents the decrease in SP-, NK1r-, nNOS-, VIP-, and S100ß-immunoreactivity (IR) and the increase in CGRP-, and CRF1r-IR. On the contrary, OB does not affect the increase in CRF2r-IR neurons observed in WRS rats and does not interfere with the mild mucosal inflammation due to WRS. Finally, OB per se increases the Mr2 expression in the muscle wall and decreases the number of the myenteric ChAT-IR neurons. Functional findings show a significantly reduction in the number of spontaneous abdominal contraction in OB treated rats. The ability of OB to block L-type Ca2+ channels, also expressed by enteric neurons, might represent a possible mechanism through which OB exerts its actions.

Loss of Interstitial Cells of Cajal and Patterns of Gastric Dysrhythmia in Patients With Chronic Unexplained Nausea and Vomiting.

BACKGROUND & AIMS: Chronic unexplained nausea and vomiting (CUNV) is a debilitating disease of unknown cause. Symptoms of CUNV substantially overlap with those of gastroparesis, therefore the diseases may share pathophysiologic features. We investigated this hypothesis by quantifying densities of interstitial cells of Cajal (ICCs) and mapping slow-wave abnormalities in patients with CUNV vs controls. METHODS: Clinical data and gastric biopsy specimens were collected from 9 consecutive patients with at least 6 months of continuous symptoms of CUNV but normal gastric emptying who were treated at the University of Mississippi Medical Center, and from 9 controls (individuals free of gastrointestinal disease or diabetes). ICCs were counted and ultrastructural analyses were performed on tissue samples. Slow-wave propagation profiles were defined by high-resolution electrical mapping (256 electrodes; 36 cm(2)). Results from patients with CUNV were compared with those of controls as well as patients with gastroparesis who were studied previously by identical methods. RESULTS: Patients with CUNV had fewer ICCs than controls (mean, 3.5 vs 5.6 bodies/field, respectively; P < .05), with mild ultrastructural abnormalities in the remaining ICCs. Slow-wave dysrhythmias were identified in all 9 subjects with CUNV vs only 1 of 9 controls. Dysrhythmias included abnormalities of initiation (stable ectopic pacemakers, unstable focal activities) and conduction (retrograde propagation, wavefront collisions, conduction blocks, and re-entry), operating across bradygastric, normal (range, 2.4-3.7 cycles/min), and tachygastric frequencies; dysrhythmias showed velocity anisotropy (mean, 3.3 mm/s longitudinal vs 7.6 mm/s circumferential; P < .01). ICCs were less depleted in patients with CUNV than in those with gastroparesis (mean, 3.5 vs 2.3 bodies/field, respectively; P < .05), but slow-wave dysrhythmias were similar between groups. CONCLUSIONS: This study defined cellular and bioelectrical abnormalities in patients with CUNV, including the identification of slow-wave re-entry. Pathophysiologic features of CUNV were observed to be similar to those of gastroparesis, indicating that they could be spectra of the same disorder. These findings offer new insights into the pathogenesis of CUNV and may help to inform future treatments.

The Telocyte Subtypes.

Several cells are endowed in the interstitial space of the connective tissue; among them, a peculiar type has been recently described and named telocyte (TC). The increasing interest on this cell type has allowed identifying it in almost all the organs. All TCs have a proper ultrastructural feature that makes them undoubtedly recognizable under the transmission electron microscope (TEM). On the contrary, a complex often confusing picture comes out from the immunohistochemical investigations either due to the technical procedures used or, intriguingly, to the possibility that diverse subtypes of TC might exist.Among the several markers used to label the TC, the most common are the CD34 and the PDGFRalpha, and, in many organs, the TC expresses both these markers. An exception is represented by the human urinary bladder where none of the TC, as recognized under the TEM, was double labelled. All the data indicate that TCs show immunohistochemical differences depending on the organ where they are located and/or the animal species.On the basis of their ubiquitous distribution, TCs are unanimously considered organizers of the connective tissue because of their ability to form 3-D networks. Close to this common role, numerous other roles have been attributed to the TC. Indeed, each of the TC subtype likely plays an own organ-/tissue-specific role contributing to different aspects of physiological regulation in the various anatomical niches they occupy.

γEpithelial Na(+) Channel (γENaC) and the Acid-Sensing Ion Channel 1 (ASIC1) expression in the urothelium of patients with neurogenic detrusor overactivity.

OBJECTIVE: To investigate the expression of two types of cation channels, γEpithelial Na(+) Channel (γENaC) and the Acid-Sensing Ion Channel 1 (ASIC1), in the urothelium of controls and in patients affected by neurogenic detrusor overactivity (NDO). In parallel, urodynamic parameters were collected and correlated to the immunohistochemical results. PATIENTS SUBJECTS AND METHODS: Four controls and 12 patients with a clinical diagnosis of NDO and suprasacral spinal cord lesion underwent urodynamic measurements and cystoscopy. Cold-cup biopsies were frozen and processed for immunohistochemistry and Western Blot. Spearman's correlation coefficient between morphological and urodynamic data was applied. One-way anova followed by Newman-Keuls multiple comparison post hoc test was applied for Western Blot results. RESULTS: In the controls, γENaC and ASIC1 were expressed in the urothelium with differences in their cell distribution and intensity. In patients with NDO, both markers showed consistent changes either in cell distribution and labelling intensity compared with the controls. A significant correlation between a higher intensity of γENaC expression in the urothelium of patients with NDO and lower values of bladder compliance was detected. CONCLUSIONS: The present findings show important changes in the expression of γENaC and ASIC1 in NDO human urothelium. Notably, while the changes in γENaC might impair the mechanosensory function of the urothelium, the increase of ASIC1 might represent an attempt to compensate for the excess in local sensitivity.

Telocytes subtypes in human urinary bladder.

Urinary bladder voiding is a complex mechanism depending upon interplay among detrusor, urothelium, sensory and motor neurons and connective tissue cells. The identity of some of the latter cells is still controversial. We presently attempted to clarify their phenotype(s) in the human urinary bladder by transmission electron microscopy (TEM) and immunohistochemistry. At this latter aim, we used CD34, PDGFRα, αSMA, c-Kit and calreticulin antibodies. Both, TEM and immunohistochemistry, showed cells that, sharing several telocyte (TC) characteristics, we identified as TC; these cells, however, differed from each other in some ultrastructural features and immunolabelling according to their location. PDGFRα/calret-positive, CD34/c-Kit-negative TC were located in the sub-urothelium and distinct in two subtypes whether, similarly to myofibroblasts, they were αSMA-positive and had attachment plaques. The sub-urothelial TC formed a mixed network with myofibroblasts and were close to numerous nerve endings, many of which nNOS-positive. A third TC subtype, PDGFRα/αSMA/c-Kit-negative, CD34/calret-positive, ultrastructurally typical, was located in the submucosa and detrusor. Briefly, in the human bladder, we found three TC subtypes. Each subtype likely forms a network building a 3-D scaffold able to follow the bladder wall distension and relaxation and avoiding anomalous wall deformation. The TC located in the sub-urothelium, a region considered a sort of sensory system for the micturition reflex, as forming a network with myofibroblasts, possessing specialized junctions with extracellular matrix and being close to nerve endings, might have a role in bladder reflexes. In conclusions, the urinary bladder contains peculiar TC able to adapt their morphology to the organ activity.

Telocytes express PDGFRα in the human gastrointestinal tract.

Telocytes (TC), a cell population located in the connective tissue of many organs of humans and laboratory mammals, are characterized by a small cell body and extremely long and thin processes. Different TC subpopulations share unique ultrastructural features, but express different markers. In the gastrointestinal (GI) tract, cells with features of TC were seen to be CD34-positive/c-kit-negative and several roles have been proposed for them. Other interstitial cell types with regulatory roles described in the gut are the c-kit-positive/CD34-negative/platelet-derived growth factor receptor α (PDGFRα)-negative interstitial cells of Cajal (ICC) and the PDGFRα-positive/c-kit-negative fibroblast-like cells (FLC). As TC display the same features and locations of the PDGFRα-positive cells, we investigated whether TC and PDGFRα-positive cells could be the same cell type. PDGFRα/CD34, PDGFRα/c-kit and CD34/c-kit double immunolabelling was performed in full-thickness specimens from human oesophagus, stomach and small and large intestines. All TC in the mucosa, submucosa and muscle coat were PDGFRα/CD34-positive. TC formed a three-dimensional network in the submucosa and in the interstitium between muscle layers, and an almost continuous layer at the submucosal borders of muscularis mucosae and circular muscle layer. Moreover, TC encircled muscle bundles, nerve structures, blood vessels, funds of gastric glands and intestinal crypts. Some TC were located within the muscle bundles, displaying the same location of ICC and running intermingled with them. ICC were c-kit-positive and CD34/PDGFRα-negative. In conclusion, in the human GI tract the TC are PDGFRα-positive and, therefore, might correspond to the FLC. We also hypothesize that in human gut, there are different TC subpopulations probably playing region-specific roles.

Glucagon-Like Peptide-2 in the Control of Gastrointestinal Motility: Physiological Implications.

Glucagon-Like Peptide-2 (GLP-2) is a pleiotropic hormone that plays several roles in different organs and tissues, so being involved in many physiological processes. Among these, it regulates gastrointestinal (GI) tract function binding to a specific G-protein coupled receptor (GLP-2R). Of note, GLP-2R is widely expressed in different cells of the GI tract, including excitatory and inhibitory neurons of the enteric nervous system. In the gut, GLP-2 has been reported to play numerous actions, among which the modulation of motility. Nevertheless, most of the GLP-2 effects and its role in physiological processes are still debated. The aim of this minireview is to summarize the data present in the literature on the control of GI motility by GLP-2, the mechanism through which it occurs, and to discuss the physiological implications of such effects. A better understanding of the role of GLP-2 on GI motor responses may be of importance for the development of new therapeutic approaches in GI dysmotility.

NK receptors, Substance P, Ano1 expression and ultrastructural features of the muscle coat in Cav-1(-/-) mouse ileum.

Caveolin (Cav)-1 is an integral membrane protein of caveolae playing a crucial role in various signal transduction pathways. Caveolae represent the sites for calcium entry and storage especially in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Cav-1(-/-) mice lack caveolae and show abnormalities in pacing and contractile activity of the small intestine. Presently, we investigated, by transmission electron microscopy (TEM) and immunohistochemistry, whether the absence of Cav-1 in Cav-1(-/-) mouse small intestine affects ICC, SMC and neuronal morphology, the expression of NK1 and NK2 receptors, and of Ano1 (also called Dog1 or TMEM16A), an essential molecule for slow wave activity in gastrointestinal muscles. ICC were also labelled with c-Kit and tachykinergic neurons with Substance P (SP). In Cav-1(-/-) mice: (i) ICC were Ano1-negative but maintained c-Kit expression, (ii) NK1 and NK2 receptor immunoreactivity was more intense and, in the SMC, mainly intracytoplasmatic, (iii) SP-immunoreactivity was significantly reduced. Under TEM: (i) ICC, SMC and telocytes lacked typical caveolae but had few and large flask-shaped vesicles we called large-sized caveolae; (ii) SMC and ICC contained an extraordinary high number of mitochondria, (iii) neurons were unchanged. To maintain intestinal motility, loss of caveolae and reduced calcium availability in Cav-1-knockout mice seem to be balanced by a highly increased number of mitochondria in ICC and SMC. Loss of Ano-1 expression, decrease of SP content and consequently overexpression of NK receptors suggest that all these molecules are Cav-1-associated proteins.

Human mesenchymal stromal cells preserve their stem features better when cultured in the Dulbecco's modified Eagle medium.

BACKGROUND AIMS: The human mesenchymal stromal cell (hMSC), a type of adult stem cell with a fibroblast-like appearance, has the potential to differentiate along the mesenchymal lineage and also along other cell lineages. These abilities make hMSC a promising candidate for use in regenerative medicine. As the hMSC represents a very rare population in vivo, in vitro expansion is necessary for any clinical use. hMSC characterization is commonly carried out through the expression of specific markers and by the capability of differentiating toward at least adipo-, osteo- and chondrocytic lineages. Commitment processes also result in significant changes in the ultrastructure in order to acquire new functional abilities; however, few studies have dealt with the ultrastructural characteristics of hMSC according to the time of incubation and type of media. METHODS: The immunophenotype, functional characteristics and ultrastructural features of bone marrow (BM) hMSC cultured in two different media were investigated. The media chosen were Iscove's modified Dulbecco's medium (IMDM) and the Dulbecco's modified Eagle medium (DMEM). The latter has been recommended recently by two international transplantation and cytotherapy societies, the International Society of Cellular Therapy (ISCT) and European Group for Blood and Bone Marrow Transplantation (EBMT), for hMSC expansion for clinical applications. RESULTS AND CONCLUSIONS: The present results indicate that culture conditions greatly influence hMSC ultrastructural features, proliferation, growth and differentiation. In particular, our findings demonstrate that DMEM preserves the hMSC stem features better. Furthermore, the results obtained in IMDM suggest that a small size does not always correlate with conditions of cell immaturity and a greater proliferative potential.