An integrated metabolomics approach using LC–HRMS, NMR and chemometrics for the profiling of the red alga Laurencia: Dereplication, tracing of bioactive compounds and detection of new natural products

Το θαλάσσιο περιβάλλον, καλύπτοντας περίπου το 70% της επιφάνειας της Γης, φιλοξενεί μια σε μεγάλο βαθμό ανεξερεύνητη βιοποικιλομορφία, προσφέροντας έτσι μια τεράστια προοπτική για την ανακάλυψη νέων βιοδραστικών ενώσεων, με στόχο τη θεραπεία ανίατων ασθενειών ή την αντιμετώπιση της ανθεκτικότητας στα φάρμακα. Προκειμένου να επιβιώσουν σε ένα διαρκώς ανταγωνιστικό οικοσύστημα, οι θαλάσσιοι οργανισμοί έχουν αναπτύξει μοναδικούς αμυντικούς μηχανισμούς, συνθέτοντας βιοδραστικές ενώσεις, οι οποίες σε ορισμένες περιπτώσεις παρουσιάζουν ασύγκριτη πολυπλοκότητα σε σχέση με τις ενώσεις που συντίθενται από τους χερσαίους οργανισμούς. Τα ροδοφύκη του γένους Laurencia θεωρούνται από τις πιο πλούσιες πηγές θαλάσσιας προέλευσης νέων δευτερογενών μεταβολιτών. Το γένος Laurencia περιλαμβάνει έως σήμερα 146 ταξινομικά αποδεκτά είδη, τα οποία συναντώνται κυρίως σε τροπικές, υποτροπικές και εύκρατες παράκτιες περιοχές, συμπεριλαμβανομένης και της Μεσογείου Θάλασσας. Παρόλο που έχει μελετηθεί εκτεταμένα τα τελευταία 50 χρόνια - οι σχετικές καταχωρήσεις στη βάση δεδομένων MarinLit υπερβαίνουν τις 1.200 - νέοι μεταβολίτες εξακολουθούν να απομονώνονται από τα είδη του γένους. Τα είδη του γένους Laurencia βιοσυνθέτουν ένα ευρύ φάσμα δευτερογενών μεταβολιτών, περιλαμβάνοντας σεσκιτερπένια, διτερπένια, τριτερπένια και C15 ακετογενίνες, οι οποίοι συχνά χαρακτηρίζονται από την παρουσία αλογόνων. Ένας μεγάλος αριθμός απομονωμένων μεταβολιτών εμφανίζει σημαντική βιολογική δράση, όπως αντιβακτηριακή, αντιμυκητιακή, εντομοκτόνο και κυτταροτοξική, όπως παρουσιάζεται από την ανασκόπηση της βιβλιογραφίας στο Κεφάλαιο 1. Πρόσφατες αναφορές εστιάζουν στην πολυπλοκότητα της ταξινόμησης των ροδοφυκών του γένους Laurencia, η οποία σχετίζεται με την εκτεταμένη μορφολογική πλαστικότητα, σε συνδυασμό με τη χημική διακύμανση, αντανακλώντας σε σημαντικό βαθμό την επίδραση περιβαλλοντικών και γενετικών παραγόντων. Η μεταβολομική αποτελεί μια αναδυόμενη τεχνολογία με εφαρμογές που εκτείνονται σε διάφορους τομείς, όπως η υγεία, τα φυσικά προϊόντα, οι βιοτεχνολογικές εφαρμογές και η διατροφή, ενώ ο αριθμός των μεταβολομικών μελετών σε θαλάσσιους οργανισμούς είναι περιορισμένος. Για μια μεταβολομική μελέτη απαιτείται η εφαρμογή αναλυτικών τεχνικών υψηλής απόδοσης, όπως η φασματοσκοπία NMR και η φασματομετρία μάζας (MS), σε συνδυασμό με εξειδικευμένα λογισμικά και βάσεις vi δεδομένων. Όσον αφορά στους θαλάσσιους οργανισμούς, ο μεγάλος αριθμός ενώσεων με άγνωστες δομές, καθώς και ο περιορισμένος αριθμός δεδομένων που μπορεί να ανακτηθεί από βάσεις δεδομένων, εξειδικευμένων στα φυσικά προϊόντα θαλάσσιας προέλευσης, αντανακλούν την αυξημένη δυσκολία στην αποτίμηση του πολύπλοκου μεταβολικού αποτυπώματος τους. Εφαρμογές της μεταβολομικής στους θαλάσσιους οργανισμούς και οι ερευνητικές προκλήσεις που σχετίζονται με αυτές εξετάζονται στο Κεφάλαιο 2. Η παρούσα μελέτη στόχευσε στο συνολικό δευτερογενές μεταβολικό αποτύπωμα εκχυλισμάτων της Laurencia εφαρμόζοντας σύγχρονες ολιστικές τεχνικές, όπως φασματοσκοπία UHPLC-PDA-HRMS και ΗR NMR, και εξειδικευμένα λογισμικά επεξεργασίας των πειραματικών δεδομένων, βάσεις δεδομένων εμπορικά διαθέσιμες (MarinLit), καθώς και in-house βιβλιοθήκες δεδομένων που αναπτύχθηκαν και εργαλεία πολυμεταβλητής ανάλυσης. Η μεθοδολογία, η οποία αναπτύχθηκε και περιγράφεται στο Κεφάλαιο 4, εφαρμόστηκε για τη σάρωση εκχυλισμάτων του γένους Laurencia ώστε να αναγνωριστούν ήδη γνωστοί βιβλιογραφικά μεταβολίτες (dereplication) και παράλληλα να εντοπιστεί η παρουσία νέων μεταβολιτών από τα πρώτα στάδια της χημικής ανάλυσης, ιεραρχώντας με αυτό τον τρόπο ποια δείγματα θα υποβάλλονταν περαιτέρω στη μακρά και επίπονη διαδικασία της φυτοχημικής ανάλυσης. Αυτή η ολοκληρωμένη προσέγγιση οδήγησε: α) Στην υλοποίηση μιας ‘in-house’ βάσης δεδομένων NMR που εστιάζει στα ροδοφύκη του γένους Laurencia και περιλαμβάνει πειραματικά ή/και βιβλιογραφικά δεδομένα NMR για δευτερογενείς μεταβολίτες προερχόμενους από είδη Laurencia και Aplysia, γνωστού και ως «λαγού της θάλασσας» που παρουσιάζει στενή οικολογική σχέση με τη Laurencia καθώς αυτή αποτελεί μέρος της διατροφής του. Η βάση δεδομένων υλοποιήθηκε στο περιβάλλον του λογισμικού ACD/Labs και διευκολύνει την αποτίμηση των δεδομένων 2D HSQC NMR με τη χρήση αλγορίθμων προσομοίωσης, οδηγώντας στην ασφαλή ταυτοποίηση των δευτερογενών μεταβολιτών που υπάρχουν στο δείγμα. Η βάση δεδομένων περιλαμβάνει περίπου 300 εγγραφές που αντιστοιχούν σε δομικά διαφορετικές ενώσεις από τις κατηγορίες των C15 ακετογενινών, σεσκιτερπενίων και διτερπενίων. β) Στην ανάπτυξη μιας βιβλιοθήκης LC-MS που περιλαμβάνει μεταβολίτες της Laurencia που έχουν απομονωθεί στο παρελθόν στο εργαστήριό μας vii γ) Στον ταχύτατο εντοπισμό δύο νέων C15 ακετογενινών της ομάδας των βρωμοαλλενίων (μαριλζαλλένιο B και χονδριοαλλένιο) σε ένα εκχύλισμα του είδους Laurencia chondrioides που συλλέχθηκε στην Κεφαλονιά. Τα αποτελέσματα της μελέτης έχουν ήδη δημοσιευθεί (Kokkotou et al., Phytochemistry, 2014, 108, 208-219). Στη συνέχεια, στο Κεφάλαιο 5, εκπονήθηκε μια μελέτη που περιλάμβανε 103 δείγματα από είδη Laurencia από τις Ελληνικές ακτές, η οποία διερεύνησε τη δυνατότητα της ανάλυσης του ολικού δευτερογενούς μεταβολικού αποτυπώματος με την εφαρμογή LCMS/PCA και 1H NMR/PCA με στόχο τη διάκριση μοτίβων μεταξύ των διαφόρων πληθυσμών του φύκους. Τα κύρια ευρήματα αυτής της μελέτης συνοψίζονται στα ακόλουθα: α) Δείγματα Laurencia, τα οποία αναδείχθηκαν ως έκτροπα (outliers) υπέδειξαν την παρουσία νέων φυσικών προϊόντων β) Aναδείχθηκαν δύο κύριοι μεταβολικοί τύποι (metabotypes) στα δείγματα Laurencia που μελετήθηκαν, ο μεταβολικός τύπος Α που περιλαμβάνει ένα φάσμα C15 ακετογενινών και ο μεταβολικός τύπος Β, που χαρακτηρίζεται κυρίως από την παρουσία σεσκιτερπενίων. γ) Επιπλέον, η μελέτη αποκάλυψε την επίδραση τη θαλάσσιας οικολογικής περιοχής (Ιόνιο και Αιγαίο Πέλαγος) σε σχέση με την έκφραση των δύο μεταβολικών τύπων στα δείγματα του είδους Laurencia obtusa. Αυτή είναι η πρώτη μεταβολομική μελέτη του ολικού δευτερογενούς μεταβολικού αποτυπώματος, η οποία πραγματοποιείται σε μεγάλο αριθμό πληθυσμών του γένους Laurencia. Στο Κεφάλαιο 6 εφαρμόστηκε στοχευμένη LC-HRMS σάρωση σε συλλογή πληθυσμών του γένους Laurencia για τον εντοπισμό βιοδραστικών διτερπενίων και συγκεκριμένα του διτερπενίου νεοροτζιολτριόλη (NRG) με ισχυρή αντιφλεγμονώδη δράση, το οποίο είχε απομονωθεί αρχικά από έναν πληθυσμό Laurencia glandulifera από την Κεφαλονιά, ενός δομικού αναλόγου της, της νεοροτζιολδιόλης, καθώς και των συγγενών δομών νεοροτζιολδιόλη Β και πρεβεζόλες Α-Ε, που είχαν απομονωθεί από ένα δείγμα L. obtusa από τις ακτές της Πρέβεζας. Αυτές οι ενώσεις πιθανόν να ανήκουν στo ίδιo βιοσυνθετικό μονοπάτι και έτσι αυτή η μελέτη σάρωσης με LC-HRMS στόχευσε στην ανίχνευση αυτών των διτερπενίων ή ακόμη και συναφών αναλόγων. Η παρουσία των ενώσεων-στόχων εντοπίστηκε σε πληθυσμό Laurencia sp. που συλλέχθηκε από τον κόλπο της Βάτσας στη Κεφαλονιά. Στη συνέχεια, η συλλογή από την ίδια περιοχή μεμονωμένων στελεχών του συγκεκριμένου πληθυσμού επέτρεψε τη viii διεξαγωγή μιας ενδο-πληθυσμιακής μελέτης. Η ανάλυση LC-HRMS/PCA κατέδειξε σαφώς την ενδο-πληθυσμιακή διακύμανση στο περιεχόμενο, αλλά ακόμα και την παρουσία/απουσία των ενώσεων-στόχων και περαιτέρω ξεχώρισε δύο ομάδες δειγμάτων συγκριτικά με τον μέσο πληθυσμό, μία ομάδα που χαρακτηρίζεται από ένα φάσμα διτερπενίων και μία άλλη ομάδα που περιλαμβάνει κυρίως σεσκιτερπένια. Στο χάρτη της PCA, η κατανομή των δειγμάτων σε σχήμα αντεστραμμένου "V", καθώς και η διευθέτηση των προαναφερθέντων ομάδων δειγμάτων υποδηλώνουν μια αντίστροφη συσχέτιση μεταξύ της αφθονίας των διτερπενίων και των σεσκιτερπενίων, αναδεικνύοντας έτσι μία «αλληλεπίδραση» (cross-talk) μεταξύ των δύο οδών που σχετίζονται με τη βιοσύνθεση των τερπενίων, δηλαδή το μονοπάτι της μεθυλερυθριτόλης (MEP) και το μονοπάτι του μεβαλονικού οξέος (MVA). Αυτή η διασύνδεση ίσως αποτελεί μια ερμηνεία για την προτιμώμενη βιοσύνθεση των βιοδραστικών διτερπενίων εις βάρος των σεσκιτερπενίων. Η μελέτη ανέδειξε επίσης την εποχιακή διακύμανση του δευτερογενούς μεταβολικού προφίλ του συγκεκριμένου πληθυσμού του ροδοφύκους, η οποία πιθανώς να εξαρτάται από τη μεταβολή των περιβαλλοντικών συνθηκών ή από την έκθεση σε παθογόνους παράγοντες, επιφυτικούς ή φυτοφάγους οργανισμούς με συνέπεια την παραγωγή βιοδραστικών διτερπενίων ή σεσκιτερπενίων.Natural products, originating from terrestrial or marine organisms, constitute a prolific source for pharmaceutically relevant products providing unique structural chemodiversity. The marine environment, covering approximately 70% of the Earth’s surface hosts a largely unexplored biodiversity, offering a huge potential for the discovery of novel compounds to target incurable diseases or overcome drug resistance. Due to the wide range of competitive ecosystems they survive in, marine organisms have developed unique defense strategies and bioactive compounds that, in some cases, are unparalleled by their terrestrial counterparts. Red algae of the genus Laurencia are considered as one of the richest sources of new secondary metabolites. The genus Laurencia currently encompasses 146 taxonomically accepted species which are mainly found in tropical, subtropical, and temperate coastal waters, including the Mediterranean Sea. Although intensively investigated for the last 50 years, new metabolites are still being isolated from Laurencia species. Displaying more than 1,200 records in MarinLit database, Laurencia species biosynthesize a wide spectrum of secondary metabolites, including sesquiterpenes, diterpenes, triterpenes and C15 acetogenins that are frequently characterized by the presence of halogen atoms. An array of the isolated metabolites exhibit important activities, such as antibacterial, antifungal, insecticidal and cytotoxic, as reviewed in Chapter 1. Recent reports comment on the complexity of the taxonomy of red algae of the genus Laurencia which is related to the extended morphological plasticity along with the chemical variation influenced to a significant degree by environmental and genetic factors. Past attempts towards species identification based on characteristic metabolites have proven unsuccessful and more importantly efforts to re-isolate bioactive compounds were severely impeded. Metabolomics consists an emerging technology with applications expanding in different areas, such as health, natural products, biotechnological applications and nutrition. Metabolomics platforms have been only applied to a limited number of projects relating to marine organisms. Metabolomics studies require the application of high-throughput analytical techniques, such as NMR spectroscopy and mass spectrometry (MS), in combination with software (s/w) tools and databases. The high number of compounds with unknown structures and the limited information retrieved from databases generated for marine organisms reflect the increased difficulty in assessing the complex ii metabolome of the marine ecosystem. Applications of metabolomics in marine organisms and research challenges are reviewed in Chapter 2. The current study addressed the global secondary metabolic profile of Laurencia crude extracts by applying state-of-the-art holistic techniques, i.e. UHPLC–PDA–HRMS and HR NMR spectroscopy, along with software tools, databases (MarinLit and those developed in-house) and multivariate data analysis tools. The developed pipeline described in Chapter 4 was applied as a screening strategy for dereplication purposes and for pinpointing the presence of new metabolites already from the very early stages of a chemical investigation and prioritize selected Laurencia crude extracts to be further subjected to the long and laborious procedure of in-depth phytochemical analysis. This integrated approach has led to: a) The construction of an ‘in-house’ Laurencia-focused NMR database, incorporating experimental and / or literature NMR data of Laurencia and Aplysia metabolites. The database is implemented in the interface of ACD/Labs s/w and facilitates the interpretation of 2D HSQC data based on simulation algorithms enhancing the confident discrimination of the secondary metabolites present in the sample. The database includes approx. 300 records comprising structurally diverse compounds from the classes of C15 acetogenins, sesquiterpenes and diterpenes. b) The development of an 'in-house’ Laurencia-focused LC-MS library comprising metabolites isolated in the past from Laurencia in our laboratory c) The rapid detection of two new C15 bromoallene acetogenins in a crude extract of Laurencia chondrioides collected from Kefalonia (marilzallene B and chondrioallene). The obtained results have been published in Kokkotou et al. Phytochemistry, 2014, 108, 208–219. Subsequently, in Chapter 5, an exploratory study comprising 103 Laurencia samples collected across the Greek coastlines investigated the potential of LC-MS/PCA and 1H NMR/PCA untargeted profiling to discriminate patterns among the various Laurencia populations. The major findings of this extended study are summarized in the following: a) The emergence of outlier samples probed to the presence of new natural products b) Two major metabotypes of Greek Laurencia specimens were revealed, metabotype A comprising an array of C15 acetogenins and metabotype B which is mainly sesquiterpenes-oriented. c) Moreover, the study revealed the influence of the marine eco-region (Ionian and Aegean Sea) relatively to the expression of the two metabotypes on Laurencia obtusa specimens. To the best of our knowledge, this is the first untargeted metabolomics study performed on a high number of Laurencia populations. In Chapter 6, a targeted LC-HRMS approach was applied to the collection of Laurencia populations screening for bioactive diterpenes and specifically the diterpene neorogioltriol (NRG) exhibiting strong anti-inflammatory activity which was initially isolated from a Laurencia glandulifera population from Kefalonia, the structural analog neorogioldiol, as well as the congeners neorogioldiol B and prevezols A-E, isolated from a Greek L. obtusa specimen from the coasts of Preveza. These compounds have been proposed to belong to the same biogenetic pathway and thus this LC-HRMS screening targeted to trace any of them or even related analogues. The presence of the target compounds was identified in a population of Laurencia collected from Vatsa bay (south-west coastal region of Kefalonia). Subsequently, collection of individual specimens of the particular Laurencia population allowed for an intra-population study. LC-MS/PCA analysis clearly revealed intra-population variability and discriminated two clusters from the average population, one associated with an array of diterpenes and another one comprising mainly sesquiterpenes. A reverse “V” shaped PCA map and the allocation of the clusters also implied an interplay between the diterpenes and sesquiterpenes abundance, suggesting a cross-talk between the two pathways related to terpenes biosynthesis, namely MEP (methylerythritol) and MVA (mevalonic acid). This cross-talk could account for the favorable biosynthesis of the bioactive diterpenes at the expense of sesquiterpenes. The seasonal variation of Laurencia secondary metabolome was also demonstrated from this study, possibly driven by the changing of environmental conditions or the exposure to pathogens, epiphytes or herbivores with an impact to the production of bioactive diterpenes or sesquiterpenes

Intestinal Upregulation of Melanin-Concentrating Hormone in TNBS-Induced Enterocolitis in Adult Zebrafish

Background: Melanin-concentrating hormone (MCH), an evolutionarily conserved appetite-regulating neuropeptide, has been recently implicated in the pathogenesis of inflammatory bowel disease (IBD). Expression of MCH is upregulated in inflamed intestinal mucosa in humans with colitis and MCH-deficient mice treated with trinitrobenzene-sulfonic acid (TNBS) develop an attenuated form of colitis compared to wild type animals. Zebrafish have emerged as a new animal model of IBD, although the majority of the reported studies concern zebrafish larvae. Regulation MCH expression in the adult zebrafish intestine remains unknown. Methods: In the present study we induced enterocolitis in adult zebrafish by intrarectal administration of TNBS. Follow-up included survival analysis, histological assessment of changes in intestinal architecture, and assessment of intestinal infiltration by myeloperoxidase positive cells and cytokine transcript levels. Results: Treatment with TNBS dose-dependently reduced fish survival. This response required the presence of an intact microbiome, since fish pre-treated with vancomycin developed less severe enterocolitis. At 6 hours post-challenge, we detected a significant influx of myeloperoxidase positive cells in the intestine and upregulation of both proinflammatory and anti-inflammatory cytokines. Most importantly, and in analogy to human IBD and TNBS-induced mouse experimental colitis, we found increased intestinal expression of MCH and its receptor in TNBS-treated zebrafish. Conclusions: Taken together these findings not only establish a model of chemically-induced experimental enterocolitis in adult zebrafish, but point to effects of MCH in intestinal inflammation that are conserved across species

Reduced Intestinal Tumorigenesis in APCmin Mice Lacking Melanin-Concentrating Hormone

Background: Melanin-concentrating hormone (MCH) is an evolutionary conserved hypothalamic neuropeptide that in mammals primarily regulates appetite and energy balance. We have recently identified a novel role for MCH in intestinal inflammation by demonstrating attenuated experimental colitis in MCH deficient mice or wild type mice treated with an anti-MCH antibody. Therefore, targeting MCH has been proposed for the treatment of inflammatory bowel disease. Given the link between chronic intestinal inflammation and colorectal cancer, in the present study we sought to investigate whether blocking MCH might have effects on intestinal tumorigenesis that are independent of inflammation. Methodology Tumor development was evaluated in MCH-deficient mice crossed to the APCmin mice which develop spontaneously intestinal adenomas. A different cohort of MCH−/− and MCH+/+ mice in the APCmin background was treated with dextran sodium sulphate (DSS) to induce inflammation-dependent colorectal tumors. In Caco2 human colorectal adenocarcinoma cells, the role of MCH on cell survival, proliferation and apoptosis was investigated. Results: APCmin mice lacking MCH developed fewer, smaller and less dysplastic tumors in the intestine and colon which at the molecular level are characterized by attenuated activation of the wnt/beta-catenin signaling pathway and increased apoptotic indices. Form a mechanistic point of view, MCH increased the survival of colonic adenocarcinoma Caco2 cells via inhibiting apoptosis, consistent with the mouse studies. Conclusion: In addition to modulating inflammation, MCH was found to promote intestinal tumorigenesis at least in part by inhibiting epithelial cell apoptosis. Thereby, blocking MCH as a therapeutic approach is expected to decrease the risk for colorectal cancer

Placebos without Deception: A Randomized Controlled Trial in Irritable Bowel Syndrome

Background: Placebo treatment can significantly influence subjective symptoms. However, it is widely believed that response to placebo requires concealment or deception. We tested whether open-label placebo (non-deceptive and non-concealed administration) is superior to a no-treatment control with matched patient-provider interactions in the treatment of irritable bowel syndrome (IBS). Methods: Two-group, randomized, controlled three week trial (August 2009-April 2010) conducted at a single academic center, involving 80 primarily female (70%) patients, mean age 47±18 with IBS diagnosed by Rome III criteria and with a score ≥150 on the IBS Symptom Severity Scale (IBS-SSS). Patients were randomized to either open-label placebo pills presented as “placebo pills made of an inert substance, like sugar pills, that have been shown in clinical studies to produce significant improvement in IBS symptoms through mind-body self-healing processes” or no-treatment controls with the same quality of interaction with providers. The primary outcome was IBS Global Improvement Scale (IBS-GIS). Secondary measures were IBS Symptom Severity Scale (IBS-SSS), IBS Adequate Relief (IBS-AR) and IBS Quality of Life (IBS-QoL). Findings: Open-label placebo produced significantly higher mean (±SD) global improvement scores (IBS-GIS) at both 11-day midpoint (5.2±1.0 vs. 4.0±1.1, p<.001) and at 21-day endpoint (5.0±1.5 vs. 3.9±1.3, p = .002). Significant results were also observed at both time points for reduced symptom severity (IBS-SSS, p = .008 and p = .03) and adequate relief (IBS-AR, p = .02 and p = .03); and a trend favoring open-label placebo was observed for quality of life (IBS-QoL) at the 21-day endpoint (p = .08). Conclusion: Placebos administered without deception may be an effective treatment for IBS. Further research is warranted in IBS, and perhaps other conditions, to elucidate whether physicians can benefit patients using placebos consistent with informed consent. Trial Registration ClinicalTrials.gov NCT0101019

Catechol-O-Methyltransferase val158met Polymorphism Predicts Placebo Effect in Irritable Bowel Syndrome

Identifying patients who are potential placebo responders has major implications for clinical practice and trial design. Catechol-O-methyltransferase (COMT), an important enzyme in dopamine catabolism plays a key role in processes associated with the placebo effect such as reward, pain, memory and learning. We hypothesized that the COMT functional val158met polymorphism, was a predictor of placebo effects and tested our hypothesis in a subset of 104 patients from a previously reported randomized controlled trial in irritable bowel syndrome (IBS). The three treatment arms from this study were: no-treatment (“waitlist”), placebo treatment alone (“limited”) and, placebo treatment “augmented” with a supportive patient-health care provider interaction. The primary outcome measure was change from baseline in IBS-Symptom Severity Scale (IBS-SSS) after three weeks of treatment. In a regression model, the number of methionine alleles in COMT val158met was linearly related to placebo response as measured by changes in IBS-SSS (p = .035). The strongest placebo response occurred in met/met homozygotes treated in the augmented placebo arm. A smaller met/met associated effect was observed with limited placebo treatment and there was no effect in the waitlist control. These data support our hypothesis that the COMT val158met polymorphism is a potential biomarker of placebo response

Hematopoietic Stem/Progenitor Cell Dependent Participation of Innate Lymphoid Cells in Low-Intensity Sterile Inflammation

Hematopoietic stem/progenitor cells (HSPC) are characterized by their unique capacities of self-renewal and multi-differentiation potential. This second property makes them able to adapt their differentiation profile depending on the local environment they reach. Taking advantage of an animal model of peritonitis, induced by injection of the TLR-2 ligand, zymosan, we sought to study the relationship between bone marrow-derived hematopoietic stem/progenitor cells (BM-HSPCs) and innate lymphoid cells (ILCs) regarding their emergence and differentiation at the site of inflammation. Our results demonstrate that the strength of the inflammatory signals affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections trigger the appearance of mature ILCs in the peritoneal cavity where the inflammation occurs. Herein, we show that only in low-dose injected mice, the recovered ILCs are dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose, the stronger inflammatory environment seems to be able to induce the emergence of ILCs independently of BM-derived HSPCs. We suggest that a relationship between HSPCs and ILCs seems to be affected by the strength of the inflammatory stimuli opening new perspectives in the manipulation of these early hematopoietic cells

MicroRNA-124 Regulates STAT3 Expression and Is Down-regulated in Colon Tissues of Pediatric Patients With Ulcerative Colitis

Background & Aims - Altered levels and functions of microRNAs (miRs) have been associated with inflammatory bowel diseases (IBDs), although little is known about their roles in pediatric IBD. We investigated whether colonic mucosal miRs are altered in children with ulcerative colitis (UC). Methods - We used a library of 316 miRs to identify those that regulate phosphorylation of STAT3 in NCM460 human colonocytes incubated with interleukin-6. Levels of miR-124 were measured by real-time PCR analysis of colon biopsies from pediatric and adult patients with UC and patients without IBD (controls), and of HCT-116 colonocytes incubated with 5-aza-2’-deoxycytidine. Methylation of the MIR124 promoter was measured by quantitative methylation-specific PCR. Results - Levels of phosphorylated STAT3 and the genes it regulates (encoding VEGF, BCL2, BCLXL, and MMP9) were increased in pediatric patients with UC, compared to control tissues. Overexpression of miR-124, let-7, miR-125, miR-26, or miR-101 reduced STAT3 phosphorylation by ≥75% in NCM460 cells; miR-124 had the greatest effect. miR-124 was downregulated specifically in colon tissues from pediatric patients with UC and directly targeted STAT3 mRNA. Levels of miR-124 were decreased whereas levels of STAT3 phosphorylation increased in colon tissues from pediatric patients with active UC, compared to those with inactive disease. Furthermore, levels of miR-124 and STAT3 were inversely correlated in mice with experimental colitis. Downregulation of miR-124 in tissues from children with UC was attributed to hypermethylation of its promoter region. Incubation of HCT-116 colonocytes with 5-aza-2’ deoxycytidine upregulated miR-124 and reduced levels of STAT3 mRNA. Conclusions - MiR-124 appears to regulate the expression of STAT3. Reduced levels of miR-124 in colon tissues of children with active UC appear to increase expression and activity of STAT3, which could promote inflammation and pathogenesis of UC in children

In vitro correlates of HIV-2-mediated HIV-1 protection

A prospective study of high-risk commercial sex workers in Senegal has shown that HIV-2 infection may reduce the risk of subsequent HIV-1 infection; these findings have been confirmed and extended, now with 13 years of observation. While exploring the biological mechanisms behind this natural protection, we found that a significant proportion of peripheral blood mononuclear cells obtained from HIV-2-infected subjects resisted in vitro challenge with CCR5-dependent HIV-1 viruses but not CXCR4-dependent viruses. High levels of beta-chemokines, the natural ligands of the CCR5 coreceptor, were correlated with low levels of viral replication, and resistance was abrogated by antibodies to beta-chemokines. Our results suggest that beta-chemokine-mediated resistance may be an important correlate of HIV protection against HIV-1 infection and relevant to HIV vaccine design

Acute Homeostatic Responses to Increased Fat Consumption in MCH1R Knockout Mice

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide which has been shown to regulate energy homeostasis. Using genetic knockout mice lacking the MCH1 receptor (MCH1R), we investigated how these mice adapt to metabolic changes caused by excessive caloric consumption. We show that the MCH system is one of the players mediating behavioral and metabolic responses upon increased caloric consumption. MCH1R knockout mice showed decreased tendency of food intake upon exposure to a high-fat diet. They also are resistant to gain weight upon high-fat diet by increasing fat metabolism. Therefore, the MCH system is important in regulating metabolic responses upon various environmental stimuli such as high-fat diet

Dissecting the Genomic Architecture of Resistance to Eimeria maxima Parasitism in the Chicken

Coccidiosis in poultry, caused by protozoan parasites of the genus Eimeria, is an intestinal disease with substantial economic impact. With the use of anticoccidial drugs under public and political pressure, and the comparatively higher cost of live-attenuated vaccines, an attractive complementary strategy for control is to breed chickens with increased resistance to Eimeria parasitism. Prior infection with Eimeria maxima leads to complete immunity against challenge with homologous strains, but only partial resistance to challenge with antigenically diverse heterologous strains. We investigate the genetic architecture of avian resistance to E. maxima primary infection and heterologous strain secondary challenge using White Leghorn populations of derived inbred lines, C.B12 and 15I, known to differ in susceptibility to the parasite. An intercross population was infected with E. maxima Houghton (H) strain, followed 3 weeks later by E. maxima Weybridge (W) strain challenge, while a backcross population received a single E. maxima W infection. The phenotypes measured were parasite replication (counting fecal oocyst output or qPCR for parasite numbers in intestinal tissue), intestinal lesion score (gross pathology, scale 0–4), and for the backcross only, serum interleukin-10 (IL-10) levels. Birds were genotyped using a high density genome-wide DNA array (600K, Affymetrix). Genome-wide association study located associations on chromosomes 1, 2, 3, and 5 following primary infection in the backcross population, and a suggestive association on chromosome 1 following heterologous E. maxima W challenge in the intercross population. This mapped several megabases away from the quantitative trait locus (QTL) linked to the backcross primary W strain infection, suggesting different underlying mechanisms for the primary- and heterologous secondary- responses. Underlying pathways for those genes located in the respective QTL for resistance to primary infection and protection against heterologous challenge were related mainly to immune response, with IL-10 signaling in the backcross primary infection being the most significant. Additionally, the identified markers associated with IL-10 levels exhibited significant additive genetic variance. We suggest this is a phenotype of interest to the outcome of challenge, being scalable in live birds and negating the requirement for single-bird cages, fecal oocyst counts, or slaughter for sampling (qPCR)