Coffee, caffeine, chlorogenic acid, and the purinergic system

Coffee is a drink prepared from roasted coffee beans and is lauded for its aroma and flavour. It is the third most popular beverage in the world. This beverage is known by its stimulant effect associated with the presence of methylxanthines. Caffeine, a purine-like molecule (1,3,7 trymetylxantine), is the most important bioactive compound in coffee, among others such as chlorogenic acid (CGA), diterpenes, and trigonelline. CGA is a phenolic acid with biological properties as antioxidant, anti-inflammatory, neuroprotector, hypolipidemic, and hypoglicemic. Purinergic system plays a key role inneuromodulation and homeostasis. Extracellular ATP, other nucleotides and adenosine are signalling molecules that act through their specific receptors, namely purinoceptors, P1 for nucleosides and P2 for nucleotides. They regulate many pathological processes, since adenosine, for instance, can limit the damage caused by ATP in the excitotoxicity from the neuronal cells. The primary purpose of this review is to discuss the effects of coffee, caffeine, and CGA on the purinergic system. This review focuses on the relationship/interplay between coffee, caffeine, CGA, and adenosine, and their effects on ectonucleotidases activities as well as on the modulation of P1 and P2 receptors from central nervous system and also in peripheral tissue

Crosstalk between the Purinergic and Immune Systems: Implications for the Glutathione Antioxidant System in Health and Disease

Glutathione (GSH) represents the major nonprotein thiol in cells and, alongside with glutathione-dependent enzymes such as glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST), exerts several biological functions including the protection against free radicals and other essential metabolic reactions within the body. Disturbances in the homeostasis of this complex glutathione antioxidant system may damage cells and have been implicated with the development and progression of several human diseases. In this context, the immune and purinergic systems are also essential, since the dysregulation in both systems may also be correlated with numerous diseases. These two networks are closely related and control inflammatory responses, especially by the crosstalk of signaling molecules, receptors, and enzymes; thus, they can exacerbate or slow down the progression of diseases. Based on this background, we aimed to provide a general scenario of the purinergic and immune systems and the connection between both and the modulation of glutathione and glutathione-dependent enzyme expression and activity in the context of health and disease

The wastewater microbiome: a novel insight for COVID-19 surveillance

Wastewater-Based Epidemiology is a tool to face and mitigate COVID-19 outbreaks by evaluating conditions in a specic community. This study aimed to analyze the microbiome proles using nanopore technology for full-length 16S rRNA sequencing in wastewater samples collected from a penitentiary (P), a residential care home (RCH), and a quarantine or health care facilities (HCF). The HCF microbiome was strongly associated with enteric bacteria previously reported in patients with chronic disease and psychological disorders. During the study, the wastewater samples from the RCH and the P were negative for SARS-CoV-2 based on qPCRs, except during the fourth week when was detected. Unexpectedly, the wastewater microbiome from RCH and P prior to week four was correlated with the samples collected from the HCF, suggesting a core bacterial community is expelled from the digest tract of individuals infected with SARS-CoV-2. We provide novel evidence that the wastewater microbiome associated with gastrointestinal manifestations appears to precede the SARS-CoV-2 detection in sewage. This nding suggests that the wastewaters microbiome can be applied as an indicator of community-wide SARS-CoV2 surveillance.This study was funded by “Fondo de Emergencia Sanitaria COVID-19, Intendencia Región de Ñuble, Chile” and FONDAP #15110027 granted by National Research and Development Agency (ANID), Chile. We also thank MINSAL and MinCiencia to support this research and provide the epidemiological data.N

Silymarin secretion and its elicitation by methyl jasmonate in cell cultures of Silybum marianum is mediated by phospholipase D-phosphatidic acid

The flavonolignan silymarin is released to the extracellular medium of Silybum marianum cultures and its production can be stimulated by the elicitor methyljasmonate (MeJA). The sequence of the signalling processes leading to this response is unknown at present. It is reported in this work that MeJA increased the activity of the enzyme phospholipase D (PLD). Treatment with mastoparan (Mst), a PLD activity stimulator, also enhanced PLD and caused a substantial increase in silymarin production. The application of the product of PLD activity, phosphatidic acid (PA) promoted silymarin accumulation. Altering PLD activity by introducing in cultures n-butanol (nBuOH), which inhibits PA production by PLD, prevented silymarin elicitation by MeJA or Mst and also impeded its release in non-elicited cultures. Treatment with iso-, sec- or tert- butanol had no effect on silymarin production. The exogenous addition of PA reversed the inhibitory action of nBuOH, both in control and MeJA-treated cultures. These results suggest that the enzyme PLD and its product PA mediate silymarin secretion to the medium of S. marianum cultures

Origin of the Metallicity Distribution in the Thick Disc

Aims. Using a suite of cosmological chemodynamical disc galaxy simulations, we assess how (a) radial metallicity gradients evolve with scaleheight; (b) the vertical metallicity gradients change through the thick disc; and (c) the vertical gradient of the stellar rotation velocity varies through the disc. We compare with the Milky Way to search for analogous trends. Methods. We analyse five simulated spiral galaxies with masses comparable to the Milky Way. The simulations span a range of star formation and energy feedback strengths and prescriptions, particle- and grid-based hydrodynamical implementations, as well as initial conditions/assembly history. Disc stars are identified initially via kinematic decomposition, with a posteriori spatial cuts providing the final sample from which radial and vertical gradients are inferred. Results. Consistently, we find that the steeper, negative, radial metallicity gradients seen in the mid-plane flatten with increasing height away from the plane. In simulations with stronger (and/or more spatially-extended) feedback, the negative radial gradients invert, becoming positive for heights in excess of !1 kpc. Such behaviour is consistent with that inferred from recent observations. Our measurements of the vertical metallicity gradients show no clear correlation with galactocentric radius, and are in good agreement with those observed in the Milky Way’s thick disc (locally). Each of the simulations presents a decline in rotational velocity with increasing height from the mid-plane, albeit the majority have shallower kinematic gradients than that of the Milky Way. Conclusions. Simulations employing stronger/more extended feedback prescriptions possess radial and vertical metallicity and kinematic gradients more in line with recent observations. The inverted, positive, radial metallicity gradients seen in the simulated thick stellar discs originate from a population of younger, more metal-rich, stars formed in-situ, superimposed upon a background population of older migrators from the inner disc; the contrast provided by the former increases radially, due to the inside-out growth of the disc. A similar behaviour may be responsible for the same flattening seen in the radial gradients with scaleheight in the Milky Way

CD83: Einblicke in dessen Funktion und transkriptioneller Regulation in humanen Tregs

Tregs play a critical role in immune homeostasis by suppressing other immune cells. Autoimmunity is a consequence of the breakdown in mechanisms controlling tolerance, which may be impacted by inadequate Treg function. Hence, an appealing therapeutic option is the modulation of Tregs. While CD83 is best known as a marker for mature dendritic cells, several other types of cells, including activated Tregs, express CD83. Previous studies suggest that CD83 is important for the regulatory capacity of Tregs. Furthermore, the tripartite transcriptional module regulating CD83 expression in DCs is cell type-specific while the regulatory elements at the CD83 locus in Tregs have not been identified. Insights into the function and transcriptional regulation of CD83 in human Tregs will be critical for potential Treg therapies in autoimmunity. To facilitate the further exploration of CD83 in human Tregs, we first honed an expansion protocol to generate sufficient stable and functional Tregs. These expanded FOXP3+ Tregs maintained CCR7/CD62L expression and upregulated markers characteristic for Tregs including Helios, LAP, as well as GARP upon stimulation. Furthermore, they were highly suppressive in vitro, in contrast to expanded CD4+ Tconvs. In-depth analyses of CD83 in these cells over 96 hours revealed that CD83 peaked as early as three hours after stimulation on mRNA and protein level, while at the later time points it was found in the soluble form in the supernatant, in line with the expression profile of non-expanded Tregs. Further investigations into the function of this molecule by knocking down its expression in Tregs revealed that CD83 does not affect the transcription factor FOXP3, crucial for Treg function, but GARP mRNA expression. GARP has not only been described as a safeguard of FOXP3, but also as a receptor for LAP in the latent TGF-β complex. Although CD83-specific siRNA transfected Tregs produced less active TGF-β upon activation, their suppressive capacity was not impacted. Finally, to determine the regulatory elements of the CD83 gene governing its expression in Tregs, ChIP-seq experiments were performed whereby four potential enhancer regions were identified based on their epigenetic profile. Further analyses revealed clusters of transcription factor binding sites for NFκB, as well as ABDB, SORY, HMTB and SORY as multiple organized regulatory elements, supporting the definition of these putative regions as enhancers. These findings offer insights into the role and transcriptional regulation of CD83 in highly stable and functional in vitro expanded Tregs. This provides the groundwork for transcriptionally targeting Tregs in vivo to modulate their function, which could lead to new clinical applications for the treatment of autoimmune diseases in the future.Tregs sind essentiell für die Aufrechterhaltung immunologischer Toleranz, da sie andere Immunzellen supprimieren. Autoimmunität ist auf eine Störung dieser Homöostase zurückzuführen, so dass eine gestörte Funktion von Tregs dazu beitragen kann. Eine attraktive therapeutische Option ist daher die Modulation der Tregs. Obwohl CD83 vor allem als Marker für reife dendritische Zellen bekannt ist, wird es auch von anderen Zelltypen, einschließlich aktivierter Tregs, exprimiert. Außerdem wird CD83 eine wichtige Rolle für die Funktionalität von murinen Tregs zugeschrieben. Während die zelltypspezifischen regulatorischen Elemente am CD83-Lokus in dendritischen Zellen schon charakterisiert wurden, sind die Mechanismen der transkriptionellen Regulation dieses Moleküls in Tregs noch unbekannt. Erkenntnisse hierüber sind jedoch für potenzielle Therapien bei Autoimmunität von sehr großem Interesse. Um die Erforschung des CD83-Moleküls in Tregs zu ermöglichen, wurde im Rahmen der vorliegenden Arbeit als erstes ein Protokoll zur Generierung einer großen Anzahl stabiler und funktionaler Tregs etabliert. Die anschließende Analyse dieser expandierten FOXP3+-Tregs zeigte eine stabile CCR7/CD62L-Expression und eine Hochregulation der für Tregs charakteristischen Marker wie Helios, LAP sowie GARP nach Stimulation. Darüber hinaus zeigten sie in vitro, im Gegensatz zu den ebenfalls expandierten konventionellen CD4+-T- Zellen, eine starke suppressorische Aktivität. Umfassende Analysen von CD83 in diesen Zellen über 96 Stunden ergaben, dass die CD83-Expression bereits drei Stunden nach der Stimulation auf mRNA- und Proteinebene seinen Höhepunkt erreichte, während es zu den späteren Zeitpunkten in der löslichen Form im Überstand vorlag, entsprechend dem Expressionsprofil der nicht-expandierten Tregs. Um die Rolle von CD83 hinsichtlich der Funktion in Tregs näher zu betrachten, wurde dessen Expression mittels RNA-Interferenz in den expandierten humanen Tregs unterdrückt. Hierbei zeigte die Herunterregulierung von CD83 keinen Einfluss auf die Expression des Transkriptionsfaktors FOXP3, welcher entscheidend für die Funktion von Tregs ist. Wir konnten allerdings eine Zunahme der Expression von GARP-mRNA detektieren, das zum einen als Stabilisator der FOXP3- Expression und zum anderen als Rezeptor für LAP im latenten TGF-β-Komplex dient. Infolge der Transfektion mit CD83-spezifischer siRNA wurde weniger aktives TGF-β durch die stimulierten Tregs sezerniert. Dies führte allerdings zu keiner verminderten suppressiven Kapazität. Um schließlich die regulatorischen Elemente des CD83-Gens zu bestimmen wurden ChIP-seq-Experimente durchgeführt. Hierbei wurden vier potenzielle Enhancer- Regionen anhand des epigenetischen Profils identifiziert. Weitere Analysen ergaben, dass Ansammlungen von Transkriptionsfaktorbindestellen für NFκB sowie ABDB, SORY, HMTB und SORY als mehrfach organisierte regulatorische Elemente vorliegen, was wiederum die Definition dieser Regionen als sogenannte Enhancer unterstützt. Die in der vorliegenden Arbeit gewonnenen Erkenntnisse unter Verwendung der hochstabilen und funktionellen in vitro expandierten Tregs geben Aufschluss über (a) die Rolle und (b) die transkriptionelle Regulation von CD83. Diese Erkenntnisse bilden die Grundlage für die zielgerichtete Expression therapeutischer Moleküle unter der Kontrolle des CD83-Promotors zur Modulation der Funktion von Tregs und damit zu neuen klinischen Anwendungen für die Behandlung von Autoimmunkrankheiten

Influence of Laminin Coating on the Autologous In Vivo Recellularization of Decellularized Vascular Protheses

Decellularization of non-autologous biological implants reduces the immune response against foreign tissue. Striving for in vivo repopulation of aortic prostheses with autologous cells, thereby improving the graft biocompatibility, we examined surface coating with laminin in a standardized rat implantation model. Detergent-decellularized aortic grafts from donor rats (n = 37) were coated with laminin and systemically implanted into Wistar rats. Uncoated implants served as controls. Implant re-colonization and remodeling were examined by scanning electron microscopy (n = 10), histology and immunohistology (n = 18). Laminin coating persisted over eight weeks. Two weeks after implantation, no relevant neoendothelium formation was observed, whereas it was covering the whole grafts after eight weeks, with a significant acceleration in the laminin group (p = 0.0048). Remarkably, the intima-to-media ratio, indicating adverse hyperplasia, was significantly diminished in the laminin group (p = 0.0149). No intergroup difference was detected in terms of medial recellularization (p = 0.2577). Alpha-smooth muscle actin-positive cells originating from the adventitial surface invaded the media in both groups to a similar extent. The amount of calcifying hydroxyapatite deposition in the intima and the media did not differ between the groups. Inflammatory cell markers (CD3 and CD68) proved negative in coated as well as uncoated decellularized implants. The coating of decellularized aortic implants with bioactive laminin caused an acceleration of the autologous recellularization and a reduction of the intima hyperplasia. Thereby, laminin coating seems to be a promising strategy to enhance the biocompatibility of tissue-engineered vascular implants