Tecniche di biologia molecolare nello studio dei sistemi chemosensoriali

TECNICHE DI BIOLOGIA MOLECOLARE NELLO STUDIO DEI SISTEMI CHEMOSENSORIALI RIASSUNTO La caratterizzazione ultrastrutturale a partire dagli anni sessanta e in seguito recentemente quella immunoistologica ha permesso di identificare cellule specializzate che possiedono diverse analogie morfologiche e molecolari con le cellule gustative ma non sono organizzate in calici gustativi. Queste cellule chemosensoriali solitarie (CCS) sono molto diffuse oltre che nella cavità orale anche nelle vie aeree e nell’apparato digerente. L’identificazione di queste cellule singole o disposte in cluster ha portato a concettualizzare l’esistenza di un sistema chemosensoriale diffuso (DCS) di cui le cellule gustative organizzate nei taste buds nella cavità orale sono solo la parte più evidente. Le funzioni del DCS sembrano coinvolgere la secrezione, la risposta immunitaria innata, l’assorbimento ma altri ruoli sono in parte ancora speculativi, e inoltre non tutti i tipi di cellule sono stati caratterizzati, lasciando aperte molte questioni che questo studio ha in parte analizzato utilizzando tecniche di biologia molecolare. In particolare sono state indagate tramite RT-PCR e western blot le differenti capacità chemorecettoriali degli organi di origine endodermica ricercando l’espressione genica dei recettori implicati nella percezione del gusto. Inoltre sono state coadiuvate ricerche finalizzate a meglio caratterizzare le cellule recettoriali all’interno dei taste buds, che risultano possedere un ventaglio di pathway molecolari molto ampio, comprendente elementi tipici delle vie aeree e dell’apparato gastrointestinale. È stato analizzato se l’espressione degli stessi geni sia modificabile a livello intestinale da diversi tipi di diete. I dati raccolti indicano una differente localizzazione dei recettori del dolce e dell’amaro lungo le vie aeree che hanno un ruolo di modulazione del movimento ciliare e della secrezione. Mentre la presenza dei recettori del dolce non va in maggior profondità della trachea, alcuni recettori dell’amaro risultano espressi anche nei polmoni. Invece lungo l’apparato gastrointestinale i recettori dell’amaro sembrano essere espressi in maniera selettiva e differenziata. L’intestino risulta inoltre sensibile a diete prolungate sovra esprimendo o sotto regolando l’espressione di proteine coinvolte nell’assorbimento e nella secrezione. In particolare è stato approfondito il sensing intestinale verso gli acidi grassi mediante quantificazione tramite PCR real time. I risultati indicano che diete iperlipidiche croniche abbattono sia l’espressione di CD36, una proteina con alta affinità per gli acidi grassi presente nella membrana apicale degli enterociti, sia l’espressione dei meccanismi di segnalazione di “saziazione” indotta dai grassi. Si tratta di un segnale, che diversamente da altri ormoni intestinali ha la capacità di prolungare la latenza tra i pasti, viene generato principalmente dalla produzione dell’ormone lipidico oleoil-etanolamide (OEA). Questo ulteriore studio oltre a evidenziare un nuovo aspetto negativo delle diete iperlipidiche, trova l’esistenza di una correlazione tra i diversi geni indagati che suggerisce che CD36 funga come sensore intestinale degli acidi grassi e abbia ruoli regolatori sui meccanismi di produzione di OEAMOLECULAR BIOLOGY TECHNIQUES IN CHEMOSENSORY SYSTEMS STUDY ABSTRACT Since 1960s, the ultrastructural and more recently the immunohystological characterization have identified specialized cells with several morphological and molecular similarities with the taste cells, without being organized into taste buds. These solitary chemosensory cells (CCS) are widespread not only in the oral cavity but even in the respiratory and gastrointestinal tract. The identification of these cell, that can be individually arranged or clustered, has led to conceptualize the existence of a diffuse chemosensory system (DCS) where the taste receptor cells organized within taste buds in the oral cavity are only the most obvious. The functions appear to involve the secretion and absorption of the DCS, and the innate immune response, but other roles are still partly speculative as not all cell types have been characterized, leaving opened many questions that this study has partly analyzed using molecular biology techniques. In particular, the different chemoreceptorial capacities of endodermal origin organs were assessed by RT-PCR and western blot examining the gene expression of those receptors that are involved in taste perception. We also performed experiments to better characterize the receptor cells within taste buds, showing that they have a very wide range of molecular pathways including typical features of the respiratory and gastrointestinal tract. It was analyzed whether the expression of these genes is modified by different types of diets at intestinal level. The data indicate a different localization of the bitter and sweet receptors along the respiratory pathway that have a role in the modulation of ciliary movement and secretion. Whereas the sweet receptors cannot be found deeper than trachea, some of the bitter receptors are also expressed in the lungs. On the other hand, along the gastrointestinal tract the bitter receptors appear to be expressed in a selective and differentiated manner. The intestine is also sensitive to prolonged diets, overexpressing or downregulating the expression of proteins involved in absorption and secretion. In particular, we studied the intestinal fatty acid sensing by real time PCR utilize. The results indicate that chronic high-fat diets cut down both the expression of CD36, a protein with high affinity for fatty acids present in the apical membrane of enterocytes, and the expression of the signaling mechanisms of satiation induced by fat. This signal, which unlike other intestinal hormones has the ability to prolong the latency between meals, is generated mainly by the production of the lipid hormone oleoylethanolamide (OEA). This study further highlights a new additional disadvantage of high-fat diets and the existence of a correlation between different genes, suggesting that CD36 act as a fatty acids sensor and have regulator roles upon the intestinal mechanisms of OEA production

Testing Homeopathy in Mouse Emotional Response Models: Pooled Data Analysis of Two Series of Studies

Two previous investigations were performed to assess the activity of Gelsemium sempervirens (Gelsemium s.) in mice, using emotional response models. These two series are pooled and analysed here. Gelsemium s. in various homeopathic centesimal dilutions/dynamizations (4C, 5C, 7C, 9C, and 30C), a placebo (solvent vehicle), and the reference drugs diazepam (1 mg/kg body weight) or buspirone (5 mg/kg body weight) were delivered intraperitoneally to groups of albino CD1 mice, and their effects on animal behaviour were assessed by the light-dark (LD) choice test and the open-field (OF) exploration test. Up to 14 separate replications were carried out in fully blind and randomised conditions. Pooled analysis demonstrated highly significant effects of Gelsemium s. 5C, 7C, and 30C on the OF parameter “time spent in central area” and of Gelsemium s. 5C, 9C, and 30C on the LD parameters “time spent in lit area” and “number of light-dark transitions,” without any sedative action or adverse effects on locomotion. This pooled data analysis confirms and reinforces the evidence that Gelsemium s. regulates emotional responses and behaviour of laboratory mice in a nonlinear fashion with dilution/dynamization

Expression of taste receptors in Solitary Chemosensory Cells of rodent airways

<p>Abstract</p> <p>Background</p> <p>Chemical irritation of airway mucosa elicits a variety of reflex responses such as coughing, apnea, and laryngeal closure. Inhaled irritants can activate either chemosensitive free nerve endings, laryngeal taste buds or solitary chemosensory cells (SCCs). The SCC population lies in the nasal respiratory epithelium, vomeronasal organ, and larynx, as well as deeper in the airway. The objective of this study is to map the distribution of SCCs within the airways and to determine the elements of the chemosensory transduction cascade expressed in these SCCs.</p> <p>Methods</p> <p>We utilized a combination of immunohistochemistry and molecular techniques (rtPCR and in situ hybridization) on rats and transgenic mice where the Tas1R3 or TRPM5 promoter drives expression of green fluorescent protein (GFP).</p> <p>Results</p> <p>Epithelial SCCs specialized for chemoreception are distributed throughout much of the respiratory tree of rodents. These cells express elements of the taste transduction cascade, including Tas1R and Tas2R receptor molecules, α-gustducin, PLCβ2 and TrpM5. The Tas2R bitter taste receptors are present throughout the entire respiratory tract. In contrast, the Tas1R sweet/umami taste receptors are expressed by numerous SCCs in the nasal cavity, but decrease in prevalence in the trachea, and are absent in the lower airways.</p> <p>Conclusions</p> <p>Elements of the taste transduction cascade including taste receptors are expressed by SCCs distributed throughout the airways. In the nasal cavity, SCCs, expressing Tas1R and Tas2R taste receptors, mediate detection of irritants and foreign substances which trigger trigeminally-mediated protective airway reflexes. Lower in the respiratory tract, similar chemosensory cells are not related to the trigeminal nerve but may still trigger local epithelial responses to irritants. In total, SCCs should be considered chemoreceptor cells that help in preventing damage to the respiratory tract caused by inhaled irritants and pathogens.</p

Exploring gastrointestinal variables affecting drug and formulation behavior: methodologies, challenges and opportunities

Various gastrointestinal (GI) factors affect drug and formulation behavior after oral administration, including GI transfer, motility, pH and GI fluid volume and composition. An in-depth understanding of these physiological and anatomical variables is critical for a continued progress in oral drug development. In this review, different methodologies (invasive versus non-invasive) to explore the impact of physiological variables on formulation behavior in the human GI tract are presented, revealing their strengths and limitations. The techniques mentioned allow for an improved understanding of the role of following GI variables: gastric emptying (magnetic resonance imaging (MRI), scintigraphy, acetaminophen absorption technique, ultrasonography, breath test, intraluminal sampling and telemetry), motility (MRI, small intestinal/colonic manometry and telemetry), GI volume changes (MRI and ultrasonography), temperature (telemetry) and intraluminal pH (intraluminal sampling and telemetry)

Extreme sensitivity of gene expression in human SH-SY5Y neurocytes to ultra-low doses of Gelsemium sempervirens.

BACKGROUND: Gelsemium sempervirens L. (Gelsemium s.) is a traditional medicinal plant, employed as an anxiolytic at ultra-low doses and animal models recently confirmed this activity. However the mechanisms by which it might operate on the nervous system are largely unknown. This work investigates the gene expression of a human neurocyte cell line treated with increasing dilutions of Gelsemium s. extract. METHODS: Starting from the crude extract, six 100 × (centesimal, c) dilutions of Gelsemium s. (2c, 3c, 4c, 5c, 9c and 30c) were prepared according to the French homeopathic pharmacopoeia. Human SH-SY5Y neuroblastoma cells were exposed for 24 h to test dilutions, and their transcriptome compared by microarray to that of cells treated with control vehicle solutions. RESULTS: Exposure to the Gelsemium s. 2c dilution (the highest dose employed, corresponding to a gelsemine concentration of 6.5 × 10(-9) M) significantly changed the expression of 56 genes, of which 49 were down-regulated and 7 were overexpressed. Several of the down-regulated genes belonged to G-protein coupled receptor signaling pathways, calcium homeostasis, inflammatory response and neuropeptide receptors. Fisher exact test, applied to the group of 49 genes down-regulated by Gelsemium s. 2c, showed that the direction of effects was significantly maintained across the treatment with high homeopathic dilutions, even though the size of the differences was distributed in a small range. CONCLUSIONS: The study shows that Gelsemium s., a medicinal plant used in traditional remedies and homeopathy, modulates a series of genes involved in neuronal function. A small, but statistically significant, response was detected even to very low doses/high dilutions (up to 30c), indicating that the human neurocyte genome is extremely sensitive to this regulation

Testing homeopathy in mouse emotional response models: pooled data analysis of two series of studies. Evid. Based Complement

Two previous investigations were performed to assess the activity of Gelsemium sempervirens (Gelsemium s.) in mice, using emotional response models. These two series are pooled and analysed here. Gelsemium s. in various homeopathic centesimal dilutions/dynamizations (4C, 5C, 7C, 9C, and 30C), a placebo (solvent vehicle), and the reference drugs diazepam (1 mg/kg body weight) or buspirone (5 mg/kg body weight) were delivered intraperitoneally to groups of albino CD1 mice, and their effects on animal behaviour were assessed by the light-dark (LD) choice test and the open-field (OF) exploration test. Up to 14 separate replications were carried out in fully blind and randomised conditions. Pooled analysis demonstrated highly significant effects of Gelsemium s. 5C, 7C, and 30C on the OF parameter &quot;time spent in central area&quot; and of Gelsemium s. 5C, 9C, and 30C on the LD parameters &quot;time spent in lit area&quot; and &quot;number of light-dark transitions,&quot; without any sedative action or adverse effects on locomotion. This pooled data analysis confirms and reinforces the evidence that Gelsemium s. regulates emotional responses and behaviour of laboratory mice in a nonlinear fashion with dilution/dynamization

Challenges in Permeability Assessment for Oral Drug Product Development

Drug permeation across the intestinal epithelium is a prerequisite for successful oral drug delivery. The increased interest in oral administration of peptides, as well as poorly soluble and poorly permeable compounds such as drugs for targeted protein degradation, have made permeability a key parameter in oral drug product development. This review describes the various in vitro, in silico and in vivo methodologies that are applied to determine drug permeability in the human gastrointestinal tract and identifies how they are applied in the different stages of drug development. The various methods used to predict, estimate or measure permeability values, ranging from in silico and in vitro methods all the way to studies in animals and humans, are discussed with regard to their advantages, limitations and applications. A special focus is put on novel techniques such as computational approaches, gut-on-chip models and human tissue-based models, where significant progress has been made in the last few years. In addition, the impact of permeability estimations on PK predictions in PBPK modeling, the degree to which excipients can affect drug permeability in clinical studies and the requirements for colonic drug absorption are addressed