Whole-Slide Image Analysis of Human Pancreas Samples to Elucidate the Immunopathogenesis of Type 1 Diabetes Using the QuPath Software

Type 1 diabetes is a chronic disease of the pancreas characterized by the loss of insulin-producing beta cells. Access to human pancreas samples for research purposes has been historically limited, restricting pathological analyses to animal models. However, intrinsic differences between animals and humans have made clinical translation very challenging. Recently, human pancreas samples have become available through several biobanks worldwide, and this has opened numerous opportunities for scientific discovery. In addition, the use of new imaging technologies has unraveled many mysteries of the human pancreas not merely in the presence of disease, but also in physiological conditions. Nowadays, multiplex immunofluorescence protocols as well as sophisticated image analysis tools can be employed. Here, we described the use of QuPath—an open-source platform for image analysis—for the investigation of human pancreas samples. We demonstrate that QuPath can be adequately used to analyze whole-slide images with the aim of identifying the islets of Langerhans and define their cellular composition as well as other basic morphological characteristics. In addition, we show that QuPath can identify immune cell populations in the exocrine tissue and islets of Langerhans, accurately localizing and quantifying immune infiltrates in the pancreas. Therefore, we present a tool and analysis pipeline that allows for the accurate characterization of the human pancreas, enabling the study of the anatomical and physiological changes underlying pancreatic diseases such as type 1 diabetes. The standardization and implementation of these analysis tools is of critical importance to understand disease pathogenesis, and may be informative for the design of new therapies aimed at preserving beta cell function and halting the inflammation caused by the immune attack

Fatty acid-related modulations of membrane fluidity in cells: detection and implications

Metabolic homeostasis of fatty acids is complex and well-regulated in all organisms. The biosynthesis of saturated fatty acids (SFA) in mammals provides substrates for ?-oxidation and ATP production. Monounsaturated fatty acids (MUFA) are products of desaturases that introduce a methylene group in cis geometry in SFA. Polyunsaturated fatty acids (n-6 and n-3 PUFA) are products of elongation and desaturation of the essential linoleic acid and ?-linolenic acid, respectively. The liver processes dietary fatty acids and exports them in lipoproteins for distribution and storage in peripheral tissues. The three types of fatty acids are integrated in membrane phospholipids and determine their biophysical properties and functions. This study was aimed at investigating effects of fatty acids on membrane biophysical properties under varying nutritional and pathological conditions, by integrating lipidomic analysis of membrane phospholipids with functional two-photon microscopy (fTPM) of cellular membranes. This approach was applied to two case studies: first, pancreatic beta-cells, to investigate hormetic and detrimental effects of lipids. Second, red blood cells extracted from a genetic mouse model defective in lipoproteins, to understand the role of lipids in hepatic diseases and metabolic syndrome and their effect on circulating cells

Μελέτη των αντιφλεγμονωδών λειτουργιών της HDL σε πειραματικά μοντέλα ποντικών

HDL has important immunomodulatory properties, including the attenuation of lipopolysaccharide (LPS)-induced inflammatory response. Τhe purpose of this study was to investigate the potential correlation between HDL shape and composition and its anti-inflammatory functions and properties in LPS-induced inflammation. As lecithin-cholesterol acyltransferase (LCAT) is a critical enzyme in the maturation of HDL we investigated whether LCAT-deficient (Lcat-/-) mice that lack mature HDL present an increased LPS-induced inflammatory response. The effects in Lcat-/- mice were compared to Apolipoprotein A-I-deficient (Apoa1-/-) mice, which lack classical HDL and are known to have an increased response to LPS, and control wild-type (WT) mice. Characterization of apolipoprotein composition of HDL, revealed that LCAT-deficient HDL is primarily composed of ApoE, while HDL from Apoa1-/- mice is highly enriched in ApoE and ApoA-II. Further analyses, showed clear differences in the lipid composition of HDL among the three groups. As expected based on the structural data of HDL, LPS (100 µg/kg body weight)-induced cytokine response in both Lcat-/- and Apoa1-/- mice was markedly enhanced and prolonged compared to WT mice. Ex vivo stimulation of whole blood with LPS (1-100 ng/mL) showed a similar enhanced pro-inflammatory phenotype. Further characterization in RAW 264.7 macrophages in vitro showed that serum and HDL, but not chylomicrons and VLDL (triglyceride-rich lipoproteins; TRL fraction) or the lipid-free protein fraction of Lcat-/- mice, had a reduced capacity to attenuate the LPS-induced TNFα response. ApoA-Ι-deficiency did not affect the capacity of HDL to neutralize LPS. Additional immunophenotyping showed that Lcat-/-, but not Apoa1-/- mice, have markedly increased circulating monocyte numbers as a result of an increase in ‘mildly pro-inflammatory’ Cd11b+LyCmid monocytes, whereas ‘highly pro-inflammatory’ Cd11b+LyChi monocytes were reduced. In line with this observation, Kuppfer cells in the Lcat-/- liver had a rather anti-inflammatory, regulatory phenotype, while peritoneal macrophages of Lcat-/- mice also showed a markedly dampened LPS-induced TNFα response. However, fluorescent microscopy studies of membrane cholesterol content and fluidity, were not able to provide a correlation between membrane cholesterol and rigidity, and macrophage responsiveness to LPS. Importantly, reintroducing LCAT by adenovirus-mediated gene transfer (AdLCAT) to Lcat-/- mice reverted their lipid profile and Ly6Chi/Ly6Cmid monocyte ratio back to that of WT mice. Consequently, AdLCAT-treated Lcat-/- mice, presented significant decrease in TNFα levels when stimulated with LPS, compared to the Lcat-/- group treated with the control adenovirus AdGFP. Based on the above, we conclude that LCAT-deficiency increases LPS-induced inflammation in mice due to reduced LPS-neutralizing capacity of immature discoidal HDL, as well as increased monocyte number despite the disturbed monocyte/macrophage phenotype.H υψηλής πυκνότητας λιποπρωτεΐνη (HDL) έχει σημαντικές ανοσορυθμιστικές ιδιότητες, συμπεριλαμβανομένης της εξασθένησης της φλεγμονώδους απόκρισης που επάγεται από τον λιποπολυσακχαρίτη (LPS). Σκοπός της παρούσας μελέτης ήταν να διερευνήσει την πιθανή συσχέτιση μεταξύ της δομής και της σύνθεσης της HDL και των αντι-φλεγμονωδών λειτουργίων της στην LPS-επαγόμενη φλεγμονή. Καθώς η λεκίθινο-χοληστερολική ακυλοτρανσφεράση (LCAT) είναι ένα κρίσιμο ένζυμο στην ωρίμανση της HDL ερευνήσαμε αν τα ποντίκια με ανεπάρκεια στο ένζυμο LCAT (Lcat-/-) που στερούνται ώριμης σφαιρικής HDL, παρουσιάζουν αυξημένη LPS-επαγόμενη φλεγμονώδη απόκριση. Η επίδραση του LPS στα Lcat-/-ποντίκια συγκρίθηκε με αυτή των ποντικιών με ανεπάρκεια στην απολιποπρωτεΐνη Α-Ι (Αροa1-/-) ποντίκια, τα οποία στερούνται κλασικής HDL και είναι γνωστό ότι έχουν αυξημένη απόκριση στο LPS, και με άγριου τύπου (WT) ποντίκια ελέγχου. Ο χαρακτηρισμός της απολιποπρωτεΐνικής σύνθεσης της HDL, αποκάλυψε ότι η HDL των Lcat-/- ποντικιών αποτελείται κατά κύριο λόγο από ΑροΕ, ενώ η HDL των Αροa1-/- ποντικιών είναι ιδιαίτερα εμπλουτισμένη σε ΑροΕ και ΑροΑ-ΙΙ. Περαιτέρω αναλύσεις έδειξαν σαφείς διαφορές στη σύνθεση των λιπιδίων της HDL μεταξύ των τριών ομάδων. Όπως αναμενόταν, η LPS-επαγόμενη (100 μg / kg βάρους σώματος) απόκριση κυτταροκινών τόσο των Lcat-/- όσο και των Αροa1-/- ποντικιών ήταν σημαντικά ενισχυμένη και παρατεταμένη σε σύγκριση με τα WT ποντίκια. Διέγερση ολικού αίματος με LPS (1-100 ng /mL) ex vivo έδειξε εναν παρόμοια ενισχυμένο προ-φλεγμονώδη φαινότυπο. Περαιτέρω χαρακτηρισμός σε RAW 264.7 μακροφάγα in vitro έδειξε ότι ο ορός και η HDL, αλλά όχι τα χυλομικρά και τα VLDL (πλούσιες σε τριγλυκερίδια λιποπρωτεΐνες - κλάσμα TRL) ή το κλάσμα απολιπιδιωμένων πρωτεΐνών των Lcat-/- ποντικιών, είχαν μειωμένη εξουδετερωτική ικανότητα της LPS-επαγόμενης απόκρισης του TNFα. Αντίθετα, η ανεπάρκεια για την ΑροΑ-Ι δεν επηρέασε την ικανότητα της HDL να εξουδετερωσει το LPS. Πρόσθετες μελέτες ανοσοφαινότυπησης έδειξαν ότι μόνο τα Lcat-/- και όχι τα Αροa1-/- ποντίκια, έχουν σημαντικά αυξημένους αριθμούς κυκλοφορούντων μονοκυττάρων, ως αποτέλεσμα της αύξησης των «ήπια προ-φλεγμονωδών» CD11b+LyCmid μονοκύτταρων, κι όχι λόγω των «έντονα προ-φλεγμονωδών» CD11b+LyChi μονοκύτταρων τα οποία παρουσίασαν αν μη τι αλλο μείωση. Σύμφωνα με αυτή την παρατήρηση, τα κύτταρα Kuppfer στο ήπαρ των Lcat-/- ποντικιών φαίνεται να έχουν έναν αντι-φλεγμονώδη, ρυθμιστικό φαινότυπο, ενώ τα περιτοναϊκά μακροφάγα των Lcat-/- έδειξαν επίσης μια αξιοσημείωτα μειωμένη LPS-επαγόμενη απόκριση του TNFα. Ωστόσο, μελέτες φθορίζουσας μικροσκοπίας έδειξαν οτι η περιεκτικότητα της μεμβράνης σε χοληστερόλη και η ρευστότητα της, δεν ήταν σε θέση να παράσχουν μία συσχέτιση μεταξύ αυτών των παραμέτρων, και της ανταπόκρισης των μακροφάγων στο LPS. Είναι σημαντικό ότι η επανεισαγωγή του ενζύμου LCAT με τη χρήση αδενοϊού (AdLCAT) στα Lcat-/- ποντίκια, επανέφερε το προφίλ λιπιδίων και την αναλογία Ly6Chi/Ly6Cmid των μονοκυττάρων στα επίπεδα των WT ποντικιών. Κατά συνέπεια, τα Lcat-/- ποντίκια στα οποία χορηγήθηκε ο αδενοϊος AdLCAT, παρουσίασαν σημαντική μείωση στα επίπεδα του TNFα μετά τη μόλυνση με LPS, σε σύγκριση με τα Lcat-/- ποντίκια που έλαβαν τον αδενοϊό ελέγχου AdGFP. Με βάση τα παραπάνω, καταλήγουμε στο συμπέρασμα ότι η ανεπάρκεια του ενζύμου LCAT στα ποντίκια, προκαλεί αύξηση της LPS-επαγόμενης φλεγμονής γεγονός που οφείλεται στη μειωμένη ικανότητα εξουδετέρωσης του LPS από την ανώριμη δισκοειδή HDL, καθώς και στον αυξημένο αριθμό των μονοκυττάρων, παρά τον διαταραγμένο φαινότυπο μονοκυττάρων/μακροφάγων

Regulation of Endothelial Nitric Oxide Synthase and High-Density Lipoprotein Quality by Estradiol in Cardiovascular Pathology

Estrogens have been recognized, in the last 3 decades, as important hormones in direct and indirect modulation of vascular health. In addition to their direct benefit on cardiovascular health, the presence of esterified estrogen in the lipid core of high-density lipoprotein (HDL) particles indirectly contributes to atheroprotection by significantly improving HDL quality and functionality. Estrogens modulate their physiological activity via genomic and nongenomic mechanisms. Genomic mechanisms are thought to be mediated directly by interaction of the hormone receptor complex with the hormone response elements that regulate gene expression. Nongenomic mechanisms are thought to occur via interaction of the estrogen with membrane-bound receptors, which rapidly activate intracellular signaling without binding of the hormone receptor complex to its hormone response elements. Estradiol in particular mediates early and late endothelial nitric oxide synthase (eNOS) activation via interaction with estrogen receptors through both nongenomic and genomic mechanisms. In the vascular system, the primary endogenous source of nitric oxide (NO) generation is eNOS. Nitric oxide primarily influences blood vessel relaxation, the heart rate, and myocyte contractility. The abnormalities in expression and/or functions of eNOS lead to the development of cardiovascular diseases, both in animals and in humans. Although considerable research efforts have been dedicated to understanding the mechanisms of action of estradiol in regulating cardiac eNOS, more research is needed to fully understand the details of such mechanisms. This review focuses on recent findings from animal and human studies on the regulation of eNOS and HDL quality by estradiol in cardiovascular pathology

Distinct Roles of Apolipoproteins A1 and E in the Modulation of High-Density Lipoprotein Composition and Function

In addition to high-density lipoprotein cholesterol (HDL-C) levels, HDL quality also appears to be very important for atheroprotection. Analysis of various clinical paradigms suggests that the lipid and apolipoprotein composition of HDL defines its size, shape, and functions and may determine its beneficial effects on human health. Previously, we reported that like apolipoprotein A-I (Apoa1), apolipoprotein E (Apoe) is also capable of promoting the <i>de novo</i> biogenesis of HDL with the participation of ATP binding cassette A lipid transporter member 1 (Abca1) and plasma enzyme lecithin:cholesterol acyltransferase (Lcat), in a manner independent of a functional Apoa1. Here, we performed a comparative analysis of the functions of these HDL subpopulations. Specifically, Apoe and Apoa1 double-deficient (<i>Apoe</i>^{<i>–/–</i>} × <i>Apoa1</i>^{<i>–/–</i>}) mice were infected with <i>APOA1-</i> or <i>APOE3-</i>expressing adenoviruses, and APOA1-containing HDL (APOA1-HDL) and APOE3-containing HDL (APOE3-HDL), respectively, were isolated and analyzed by biochemical and physicochemical methods. Western blot and lipidomic analyses indicated significant differences in the apolipoprotein and lipid composition of the two HDL species. Moreover APOE3-HDL presented a markedly reduced antioxidant potential and Abcg1-mediated cholesterol efflux capacity. Surprisingly, APOE3-HDL but not APOA1-HDL attenuated LPS-induced production of TNFα in RAW264.7 cells, suggesting that the anti-inflammatory effects of APOA1 are dependent on APOE expression. Taken together, our data indicate that APOA1 and APOE3 recruit different apolipoproteins and lipids on the HDL particle, leading to structurally and functionally distinct HDL subpopulations. The distinct role of these two apolipoproteins in the modulation of HDL functionality may pave the way toward the development of novel pharmaceuticals that aim to improve HDL functionality