Study of the effect of nutritional and endogenous factors on adipocyte differentiation: the role of anandamide

Obesity is a complex metabolic disorder originating from an imbalance between energy intake and expenditure. Obesity is attributed to the number and size of adipocytes, which are the main cells of the adipose tissue. Mature adipocytes have a spherical shape and contain accumulated triglycerides in the form of a large oil droplet. They are derived from preadipocytes, fibroblast-like cells, through the differentiation process. Several endogenous and exogenous factors affect this process which, apart from triglyceride accumulation, is accompanied by the appearance of early, intermediate and late mRNA/protein markers. Increasing evidence supports the hypothesis that the endocannabinoid system (ES) holds an important regulatory role on the energy homeostasis and consequently on obesity, through central and peripheral mechanisms. The most studied endocannabinoid is anandamide (AEA), which increases food intake and promotes weight gain in animals by activating the cannabinoid 1(CB1) receptor. The selective inhibition of CB1 receptor with Rimonabant (SR141716A), on the contrary, reduces food intake and body weight in obese animals and humans. The aim of this study was to investigate the involvement of AEA in the differentiation process of rat adipocytes using primary cell cultures of rat preadipocytes. We showed that AEA increases preadipocyte differentiation into adipocytes in a concentration- and time-dependent manner. In addition, we found that PPARγ2 gene expression was increased during the differentiation process induced by AEA. Moreover, our results showed that CB1 and CB2 receptors were also expressed in differentiated adipocytes. Furthermore, CB1 receptor expression level was nearly doubled in differentiated adipocytes in the presence of AEA, while CB2 receptor gene expression and protein expression was attenuated. The effect of the hydrolysis-resistant analogue of AEA, (R+)-mAEA, on the differentiation process was also studied in order to clarify if arachidonic acid (AA), derived from AEA hydrolysis by fatty acid amide hydrolase (FAAH), could be involved in the induction of the differentiation. The RNA and the protein levels of FAAH were upregulated in differentiated adipocytes by AEA. On the contrary, R(+)-mAEA had a lower effect on adipocyte differentiation in comparison to AEA, suggesting that AA is involved in this process. In addition, URB597, a specific FAAH inhibitor, decreases the differentiation not only in the presence but also in the absence of AEA. Our findings suggest that cycloxygenase 2 (COX-2) is present in rat adipose tissue. These findings, in combination with the fact that AEA is a good substrate for COX-2, led us to further investigate the effect of AEA on COX-2 gene expression during the differentiation process in the presence or absence of indomethacin (IND), a pan-COX inhibitor. COX-2 gene expression was higher in cells treated with AEA for 7 days compared with untreated cells. In addition, IND reduced differentiation in the absence or presence of AEA and although the reduction was not statistically significant, we observed a strong inhibitory effect. These results suggest that COX-2 derivatives of AEA and/or AA are involved on adipocyte differentiation. ........................................................................................................................................Η παχυσαρκία είναι αποτέλεσμα της μακροχρόνιας διατήρησης του οργανισμού σε κατάσταση θετικού ενεργειακού ισοζυγίου και κατατάσσεται σε δυο κύριες κατηγορίες, την υπερτροφική και την υπερπλαστική παχυσαρκία. Η υπερτροφική παχυσαρκία χαρακτηρίζεται από την αύξηση του όγκου των λιποκυττάρων ενώ η υπερπλαστική από την αύξηση του αριθμού τους. Ο αριθμός των λιποκυττάρων αυξάνει κυρίως μέσω της αύξησης της ταχύτητας της διαφοροποίησης των προλιποκυττάρων σε λιποκύτταρα. Τα προλιποκύτταρα είναι εξειδικευμένοι ινοβλάστες ενώ τα λιποκύτταρα έχουν στρογγυλό σχήμα και το εσωτερικό τους καταλαμβάνεται σχεδόν εξ’ ολοκλήρου από μια λιποσταγόνα συσσωρευμένων TG και κατά την πορεία της διαφοροποίησης συμμετέχουν, όπως έχει αποδειχθεί, πολλοί περιβαλλοντικοί αλλά και γενετικοί παράγοντες. Τα τελευταία χρόνια, πολλές είναι οι βιβλιογραφικές αναφορές που προτείνουν έναν κεντρικό ρόλο του ES στην ενεργειακή ομοιόσταση. Το AEA, ενδογενές παράγωγο του ΑΑ, αποτελεί το σημαντικότερο μέλος των ενδοκανναβινοειδών. Στην παρούσα εργασία αποδείχτηκε ότι το ΑΕΑ επάγει τη πορεία της διαφοροποίησης των προλιποκυττάρων αρουραίων, με δοσο- και χρονοεξαρτώμενο τρόπο, και προκαλεί την αύξηση της έκφρασης του μεταγραφικού παράγοντα PPARγ2. Επίσης, αποδείχτηκε η παρουσία του ES στα διαφοροποιημένα λιποκύτταρα αρουραίου και η μεταβολή της έκφρασης των συστατικών του συστήματος αυτού κατά την πορεία της διαφοροποίησης, παρουσία του ΑΕΑ. Το ΑΕΑ επάγει τη διαφοροποίηση εν μέρει μέσω μεταβολισμού του σε ΑΑ, όπως φάνηκε από τα αποτελέσματα μελέτης της επίδρασης του σταθερού σε υδρόλυση αναλόγου του ΑΕΑ, R(+)-mAEA, και του ειδικού αναστολέα της FAAH, URB597, στην πορεία της διαφοροποίησης. Στη προσπάθεια μας να διευκρινίσουμε περαιτέρω το μηχανισμό δράσης του ΑΕΑ στη διαφοροποίηση, διερευνήσαμε την ύπαρξη της COX-2, στα διαφοροποιημένα λιποκύτταρα αρουραίου. Τα αποτελέσματά μας έδειξαν ότι η COX-2 εκφράζεται στα κύτταρα αυτά, με την έκφραση του γονιδίου του ενζύμου, να είναι αυξημένη στα διαφοροποιημένα λιποκύτταρα, παρουσία του ΑΕΑ. Το γεγονός αυτό αναδεικνύει την εμπλοκή της COX-2 στη δράση του ΑΕΑ στη πορεία της διαφοροποίησης. Η περιοχή που εντοπίζεται ο λιπώδης ιστός, η παροχή μακρο- και μικροθρεπτικών συστατικών, η παρουσία άλλων κυτταρικών τύπων συνεισφέρουν στη διαφορετικότητα της μορφολογίας, της λειτουργίας και της εμπλοκής κάθε τύπου λιπώδους ιστού στην ανάπτυξη της παχυσαρκίας. Το γεγονός αυτό σε συνδυασμό με τα αποτελέσματά μας βάσει των οποίων διαπιστώθηκε η ύπαρξη του ES στο λιπώδη ιστό αρουραίου, μας οδήγησε στη διερεύνηση της ύπαρξης του ES σε διάφορους τύπους ανθρώπινου λιπώδους ιστού και συγκεκριμένα στον υποδόριο κοιλιακό, στο γαστρικό και στο μεσεντερικό λιπώδη ιστό παχύσαρκων εθελοντών, καθώς επίσης και στη μελέτη της επίδρασης του ΑΕΑ στη διαφοροποίηση προλιποκυττάρων, που απομονώθηκαν από τους προαναφερθέντες τύπους ιστών. Παράλληλα, μελετήθηκε η επίδραση της IBMX στη διαφοροποίηση των ανθρώπινων προλιποκυττάρων. Αρχικά, φάνηκε ότι παρουσία της ΙΒΜΧ είναι απαραίτητη για τη διαφοροποίηση των ανθρώπινων προλιποκυττάρων, αλλά και απαραίτητη για την επίδραση του ΑΕΑ στην πορεία της διαφοροποίησης. ....................................................................................................

Heterodimeric IL-15 in Cancer Immunotherapy

Immunotherapy has emerged as a valuable strategy for the treatment of many cancer types. Interleukin-15 (IL-15) promotes the growth and function of cytotoxic CD8+ T and natural killer (NK) cells. It also enhances leukocyte trafficking and stimulates tumor-infiltrating lymphocytes expansion and activity. Bioactive IL-15 is produced in the body as a heterodimeric cytokine, comprising the IL-15 and the so-called IL-15 receptor alpha chain that are together termed “heterodimeric IL-15” (hetIL-15). hetIL-15, closely resembling the natural form of the cytokine produced in vivo, and IL-15:IL-15Rα complex variants, such as hetIL-15Fc, N-803 and RLI, are the currently available IL-15 agents. These molecules have showed favorable pharmacokinetics and biological function in vivo in comparison to single-chain recombinant IL-15. Preclinical animal studies have supported their anti-tumor activity, suggesting IL-15 as a general method to convert “cold” tumors into “hot”, by promoting tumor lymphocyte infiltration. In clinical trials, IL-15-based therapies are overall well-tolerated and result in the expansion and activation of NK and memory CD8+ T cells. Combinations with other immunotherapies are being investigated to improve the anti-tumor efficacy of IL-15 agents in the clinic

Genome-Wide Analysis of lncRNA-mRNA Co-Expression Networks in CD133+/CD44+ Stem-like PDAC Cells

Pancreatic ductal adenocarcinoma (PDAC), the second most prevalent gastrointestinal malignancy and the most common type of pancreatic cancer is linked with poor prognosis and, eventually, with high mortality rates. Early detection is seldom, while tumor heterogeneity and microarchitectural alterations benefit PDAC resistance to conventional therapeutics. Although emerging evidence suggest the core role of cancer stem cells (CSCs) in PDAC aggressiveness, unique stem signatures are poorly available, thus limiting the efforts of anti-CSC-targeted therapy. Herein, we report the findings of the first genome-wide analyses of mRNA/lncRNA transcriptome profiling and co-expression networks in PDAC cell line-derived CD133+/CD44+ cells, which were shown to bear a CSC-like phenotype in vitro and in vivo. Compared to CD133−/CD44− cells, the CD133+/CD44+ population demonstrated significant expression differences in both transcript pools. Using emerging bioinformatic tools, we performed lncRNA target coding gene prediction analysis, which revealed significant Gene Ontology (GO), pathway, and network enrichments in many dyregulated lncRNA nearby (cis or trans) mRNAs, with reported involvement in the regulation of CSC phenotype and functions. In this context, the construction of lncRNA/mRNA networks by ingenuity platforms identified the lncRNAs ATF2, CHEK1, DCAF8, and PAX8 to interact with “hub” SC-associated mRNAs. In addition, the expressions of the above lncRNAs retrieved by TCGA-normalized RNAseq gene expression data of PAAD were significantly correlated with clinicopathological features of PDAC, including tumor grade and stage, nodal metastasis, and overall survival. Overall, our findings shed light on the identification of CSC-specific lncRNA signatures with potential prognostic and therapeutic significance in PDAC

Tumor eradication by hetIL-15 locoregional therapy correlates with an induced intratumoral CD103intCD11b+ dendritic cell population

Summary: Locoregional monotherapy with heterodimeric interleukin (IL)-15 (hetIL-15) in a triple-negative breast cancer (TNBC) orthotopic mouse model resulted in tumor eradication in 40% of treated mice, reduction of metastasis, and induction of immunological memory against breast cancer cells. hetIL-15 re-shaped the tumor microenvironment by promoting the intratumoral accumulation of cytotoxic lymphocytes, conventional type 1 dendritic cells (cDC1s), and a dendritic cell (DC) population expressing both CD103 and CD11b markers. These CD103intCD11b+DCs share phenotypic and gene expression characteristics with both cDC1s and cDC2s, have transcriptomic profiles more similar to monocyte-derived DCs (moDCs), and correlate with tumor regression. Therefore, hetIL-15, a cytokine directly affecting lymphocytes and inducing cytotoxic cells, also has an indirect rapid and significant effect on the recruitment of myeloid cells, initiating a cascade for tumor elimination through innate and adoptive immune mechanisms. The intratumoral CD103intCD11b+DC population induced by hetIL-15 may be targeted for the development of additional cancer immunotherapy approaches

CRH Promotes the Neurogenic Activity of Neural Stem Cells in the Adult Hippocampus

Local cues in the adult neurogenic niches dynamically regulate homeostasis in neural stem cells, whereas their identity and associated molecular mechanisms remain poorly understood. Here, we show that corticotropin-releasing hormone (CRH), the major mediator of mammalian stress response and a key neuromodulator in the adult brain, is necessary for hippocampal neural stem cell (hiNSC) activity under physiological conditions. In particular, we demonstrate functionality of the CRH/CRH receptor (CRHR) system in mouse hiNSCs and conserved expression in humans. Most important, we show that genetic deficiency of CRH impairs hippocampal neurogenesis, affects spatial memory, and compromises hiNSCs' responsiveness to environmental stimuli. These deficits have been partially restored by virus-mediated CRH expression. Additionally, we provide evidence that local disruption of the CRH/CRHR system reduces neurogenesis, while exposure of adult hiNSCs to CRH promotes neurogenic activity via BMP4 suppression. Our findings suggest a critical role of CRH in adult neurogenesis, independently of its stress-related systemic function

Systemic IL-15, IFN-gamma, and IP-10/CXCL10 signature associated with effective immune response to SARS-CoV-2 in BNT162b2 mRNA vaccine recipients

Early responses to vaccination are important for shaping both humoral and cellular protective immunity. Dissecting innate vaccine signatures may predict immunogenicity to help optimize the efficacy of mRNA and other vaccine strategies. Here, we characterize the cytokine and chemokine responses to the 1st and 2nd dose of the BNT162b2 mRNA (Pfizer/BioNtech) vaccine in antigen-naive and in previously coronavirus disease 2019 (COVID-19)-infected individuals (NCT04743388). Transient increases in interleukin-15 (IL-15) and interferon gamma (IFN-gamma) levels early after boost correlate with Spike antibody levels, supporting their use as biomarkers of effective humoral immunity development in response to vaccination. We identify a systemic signature including increases in IL-15, IFN-gamma, and IP-10/CXCL10 after the 1st vaccination, which were enriched by tumor necrosis factor alpha (TNF-alpha) and IL-6 after the 2nd vaccination. In previously COVID-19-infected individuals, a single vaccination results in both strong cytokine induction and antibody titers similar to the ones observed upon booster vaccination in antigen-naive individuals, a result with potential implication for future public health recommendations