Monocytes/macrophages have begun to emerge as key cellular modulators of brain homeostasis and central nervous system (CNS) disease. In the healthy brain, resident microglia are the predominant macrophage cell population; however, under conditions of blood-brain barrier leakage, peripheral monocytes/macrophages can infiltrate the brain and participate in CNS disease pathogenesis. Distinguishing these two populations is often challenging, owing to a paucity of universally accepted and reliable markers. To identify discriminatory marker sets for microglia and peripheral monocytes/macrophages, we employed a large meta-analytic approach using five published murine transcriptional datasets. Following hierarchical clustering, we filtered the top differentially expressed genes (DEGs) through a brain cell type-specific sequencing database, which led to the identification of eight microglia and eight peripheral monocyte/macrophage markers. We then validated their differential expression, leveraging a published single cell RNA sequencing dataset and quantitative RT-PCR using freshly isolated microglia and peripheral monocytes/macrophages from two different mouse strains. We further verified the translation of these DEGs at the protein level. As top microglia DEGs, we identified P2ry12, Tmem119, Slc2a5 and Fcrls, whereas Emilin2, Gda, Hp and Sell emerged as the best DEGs for identifying peripheral monocytes/ macrophages. Lastly, we evaluated their utility in discriminating monocyte/macrophage populations in the setting of brain pathology (glioma), and found that these DEG sets distinguished glioma-associatedmicroglia from macrophages in both RCAS and GL261 mouse models of glioblastoma. Taken together, this unbiased bioinformatic approach facilitated the discovery of a robust set of microglia and peripheral monocyte/macrophage expression markers to discriminate these monocyte populations in both health and disease.
The resident macrophages of the brain are termed microglia. Those cells localize to brain during early development and form the ramified microglia thereafter [Hanisch and Kettenmann, 2007]. The activity of microglia depends on the type of pathology. In glioma microenvironment, microglia are thought to be inactive due to the immunosuppressive cytokines secreted by GBM, such as IL-10, IL-6, IL-4, TGF-β and Prostaglandin E2 [Wei et al, 2010]. Additionally, microglia express low levels of MHC Class II molecule along with some other costimulatory molecules [Badie et al, 2002]. When stimulated with lipopolysaccharides (LPS) and IFN-γ microglia assume M1 phenotype to secrete pro-inflammatory cytokines such as TNF-α, IL-1β and IL-12, present antigen and express high levels of inducible NO (iNOS) for NO production. This phenomenon takes place to kill pathogens and induce T cells for adaptive immune response [Gordon and Taylor, 2005]. In addition to M1 phenotype, microglia (and macrophages) display an M2 phenotype where they express anti-inflammatory cytokines such as IL-4, IL-10, IL3 and TGF-β, as well as Arginase-1 (Arg1) and CD206 which then leads to allergy response, parasite clearance, inflammatory dampening, tissue remodeling, angiogenesis, immune regulation and tumor promotion [Villalta et al., 2009]. In this respect, microglial cells seem to acquire M2 phenotype with the increasing histological malignancy. M2 phenotype represents the homeostatic state while M1 phenotype is a sign of inflammation. Given these facts, in GBM microenvironment, microglia assume an activated morphology but rather a different phenotype from that of a regular inflammation [Komohara et al, 2008; Charles et al, 2011]. Yet, microglia mediate tumor cell migration and tumor growth via MT1-MMP secretion in response to cues released from glioma cells, a phenomenon observed only when microglia are in the glioma-induced state [Markovic et al, 2005; Sliwa et al, 2007]. However, there are two contradictory studies where depletion of microglia, by Markovic et al, resulted in 80% decrease in tumor volume, while macrophage depletion, by Gallernau et al, induced 33% increase in the tumor volume, showing that myeloid depletion is both pro- and anti- tumorigenic due to potential unaccounted targeting of additional factors [Markovic et al, 2009; Galarneau et al, 2007].
Like other solid cancers, gliomas are heterogeneous and keep interacting with other “healthy” resident cells in the brain. Gliomas may not only derive from the healthy cells but after the initiation they start to influence these cells and convert them to a tumor supporting phenotype. Among different types of tumor associated parenchymal cell populations in the brain, gliomaassociatedmicroglia/macrophages would be the most interesting subtype. Firstly, they shared a large proportion of gliomas with a contribution of up to 30% of tumor mass. Secondly, as the guardians in the brain, microglial cells constantly screen brain tissue using their motile processes, once an insult is found, they become activated and move to the lesions very rapidly. However, this property raises a few interesting questions: How do microglial cells behave when brain resident cells just start to transform to tumor cells? Do they sense these events? Do they accumulate around it? Do they phagocytose these transformed cells? These questions are still obscure. Last but not least, microglial cells are the immune cells of the brain, they express a wide range of receptors like neurotransmitter receptors, pattern-recognition receptors and cytokine and chemokine receptors (Kettenmann et al. 2011), by which they may easily get activated by tumor released factors or physical contact. And they could also release different types of cytokines and chemokines, which may shape the characteristics of gliomas. Glioma research has been extensively developed in the past decades; however, the majority mainly focus on the biology of the tumor itself without much concern about tumor microenvironment. These tumor-associated resident brain cells also contribute immensely to gliomagenesis, and more importantly, these cells may also play part in drug resistance in conventional chemotherapy.
Keywords: liver diseases, macrophage phenotypes, therapeutic targets, treatment strategies, innate immunity
Editorial on the Research Topic Macrophages in Liver Disease
Macrophages constitute a key component of our immune system and play an important role in immune surveillance. Hepatic macrophages are a heterogeneous population of immune cells that mainly comprises of embryonically-derived resident Kupffer cells (KCs), and circulating monocyte-derived macrophages (MoMFs). They play a critical role in disease initiation and progression as well as contribute to disease resolution. Traditionally, macrophages were defined by two broad subsets: classically-activated pro-inflammatory M1 or alternatively-activated anti-inflammatory M2 macrophages. However, it has been recognized that macrophages can differentiate into multiple phenotypes with distinct functions based on the tissue microenvironment.
Chronic neuroinflammation is a common feature in neurological diseases such as multiple sclerosis, Huntington’s, Parkinson’s, Alzheimer’s and stroke, and since microglia are the resident immune cells of the CNS, their activation has been implicated in the progression of a number of diseases. Microglia can be activated by a vast number of substances including many proteins associated with neurodegenerative disease such as amyloid-beta (Jana et al., 2008), alpha- synuclien (Zhang et al., 2005) and prions (Szpak et al., 2006), as well as through injury (Maeda et al., 2010), ischemia (Hur et al., 2010) and infection (Xu et al., 2009). Activation of microglia does not always lead to detrimental effects. During traumatic injury, microglia have been shown to clear glutamate, whose excessive release results in excititoxicity in stroke, ALS and autism among others, without evoking inflammatory mediators (Shaked et al., 2005). During axotomy of the optic nerve, microglia have also been shown to efficiently clear myelin debris (Battisti et al., 1995). In this study we have focused on the expression of various receptors on microglia from mouse models of glioma, multiple sclerosis, Alzheimer’s disease and stroke. The function and behaviour of microglia in these diseases is summarised below.
All MT-MMPs enhanced the migration of astrocytes in the OPoSSM assay. The most potent mediators of astrocyte migration were MMP14, -17, and -25. Interestingly, MMP14 showed higher numbers of migrating astrocytes than the control in only the first two millimeters from the edge of the slice, while the GPI-anchored MMP17 and MMP25 had the maximum migration-enhancing effect on astrocytes with regard to both chain number and penetration depth. Since MT-MMPs have different substrate specificities and activities, these different effects on migration might be due to a direct effect of a particular MT-MMP on the migration of astrocytes, or an indirect effect that is mediated by downstream factors that are activated by this particular MT-MMP [ 40 ]. A likely candidate would be MMP2, because this gelatinase is activated by most of the MT-MMPs and can enhance the migration of different cell types, such as 3T3 fibroblasts and glioma cells [ 18 , 29 , 41 ]. The astrocytes used in our model showed mostly activated MMP2 in culture supernatants, and we did not observe further activation by any of the MT-MMPs. Therefore, it is unlikely that active MMP2 mediated the MT-MMP-induced enhancement of astrocyte migration in our migration and invasion models. One could rather argue that the high amount of active MMP2 secreted by the astrocytes used here led to the constitutively high migration potential of these cells, thereby dwarfing the effect of the transduced MT-MMPs. It is not clear why the GPI-anchored MMP17 and MMP25 had the strongest effect on astrocyte migration. MMP17 digests ECM substrates such as gelatin and is, among others, an activator of proTNF and of ADAMTS4 [ 21 ]. Active ADAMTS4 digests several ECM constituents including the brain-specific brevican, and enables brain invasion of 9L rat gliosarcoma cells [ 42 ]. This mechanism could be in action in our model and might be relevant for human disorders, since ADAMTS4 is expressed in human glioblastoma [ 43 ].
Vorherige Inkubationsexperimente im OT haben gezeigt, daß die Autofluoreszenz der Zellen vernachlässigbar klein gegenüber der Fluoreszenz des Farbstoffs ist, und daß 5 µM Farbstoff im Nährmedium bei 37 °C und 45 min Inkubationszeit optimale Bedingungen für die Aufnahme der Acetoxymethylesterderivate von SNARF-1 für C6-Glioma-Zellen sind. Inhomogene Färbungen der verschiedenen Zellbereiche treten bei SNARF-1 häufig noch im Verlauf des Experiments auf, geringere Temperaturen können diese Kompartimentierungen verlangsamen, führen aber gleichzeitig zu unphysiologischen Meßbedingungen (s. Anhang A.VI). Deshalb und um die Vergleichbarkeit mit anderen Experimenten zu gewährleisten wurden alle Experimente bei 37°C durchgeführt. Selbiges gilt für Fluo-4 (in situ Eichung von BCECF und zur Fura-2-Kalibration s. Anhang A.IIc und IV).
Microglia significantly contribute to the glioma tumor mass by infiltrating primary tumor mass (Fig 5.1. A). The net effect of microglial abundance in gliomas is tumor promoting by inducing the glioma invasiveness (Fig 5.1. B). The result of microglia- glioma cross-talk is escalation of MMP-2 activation and that leads to increased brakedown of ECM proteins which can explain increase of glioma invasiveness (Fig 5.1. C-D). Glioma release a factor which stimulates the production of a major MMP-2 activator- MT1-MMP in microglia. Moreover, the expression of MT1-MMP is mediated by p38 MAPK, which makes this kinase a possible target for therapy of invasive gliomas (Fig 5.1. D).
The “5-to-7-year shift” refers to the remarkable improvements observed in children’s cognitive abilities during this age range, particularly in their ability to exert control over their attention and behavior—that is, their executive functioning. As this shift coincides with school entry, the extent to which it is driven by brain maturation or by exposure to formal schooling is unclear. In this longitudinal study, we followed 5-year-olds born close to the official cutoff date for entry into first grade and compared those who subsequently entered first grade that year with those who remained in kindergarten, which is more play oriented. The first graders made larger improvements in accuracy on an executive- function test over the year than did the kindergartners. In an independent functional MRI task, we found that the first graders, compared with the kindergartners, exhibited a greater increase in activation of right posterior parietal cortex, a region previously implicated in sustained attention; increased activation in this region was correlated with the improvement in accuracy. These results reveal how the environmental context of formal schooling shapes brain mechanisms underlying improved focus on cognitively demanding tasks.
The most negatively charged p h o sp h o lip id s PS and PIN, although they are in sm all am o u n ts in SPM , are involved in im p o rta n t p h ysiological processes in the central nervous system . P IN lipids are well know n to be involved in sy n a p tic tra n s mission and their catab o lism plays an im p o rta n t and perhaps prim ary central role in th e b io c h em ica l events associated w ith physiological activity [2, 3]. Intravenous injection o f a sonicated d isp e rsio n o f brain phospholipids results in a sig n ifican t increase o f both the d o pam ine-sensitive a d e n y la te cyclase activity, and the cyclic A M P content o f m o u se b ra in , U nder these experim ental cond itio n s, PS lip o somes stim ulate the release o f acety lch o lin e from ra t cerebral cortex  and th e release o f h ista m in e from mast-cells , M oreover, PS vesicles (liposom es)
Even though the negatively charged phospho lipids PS and PIN occur in m inute am ounts in SPM , they cause im portant physiological processes in the central nervous system. PIN lipids are well known to be involved in synaptic transm ission and their catabolism plays an im portant and perhaps primary central role in the biochem ical events as sociated with the physiological activity [2, 3]. Intra venous injection of a sonicated dispersion of brain phospholipids results in a significant increase both the dopamine-sensitive adenylate cyclase activity, and the cyclic AMP content of mouse brain . Under these experimental conditions, PS liposomes increase in the metabolism of catecholam ines in the brain of animals , stim ulate the release of acetyl choline from rat cerebral cortex and the release of histamine from mast cells [6, 7], M oreover, PS vesicles (liposomes) interact with biological m em branes causing changes of the m em brane physico chemical properties . Such alterations of the state in the bilayer can modify the activities o f m em brane-bound enzymes, e.g. N a+, K+-ATPase, acetyl cholinesterase, C a2+-stim ulated ATPase and ade nylate cyclase [9-11]. Changes in the viscosity of
A fundamental question is the mechanism by which reduced BECN1 levels and autophagy modulate the IL-1beta/IL-18 produc- tion. Proposed mechanisms include degradation of pro-IL-1beta (Harris et al, 2011), degradation of inflammasomes (Shi et al, 2012), and an indirect activation of the inflammasome via accumu- lation of dysfunctional mitochondria and oxidative stress due to impaired mitophagy (Saitoh et al, 2008; Zhou et al, 2011; Lodder et al, 2015; Lee et al, 2016; Ye et al, 2017). Since no significant changes in pro-IL-1beta levels were observed, we investigated other steps in the IL-1beta/IL-18 processing pathway. Here, increased levels of NLRP3 and cleaved CASP1 alongside a higher percentage of cells with ASC-stained inflammasomes were detected. Pro-CASP1 is cleaved into p10 and p20 fragments, which were reported to be degraded via the ubiquitin–proteasome system (Squires et al, 2007; Muehlbauer et al, 2010; Van Opdenbosch et al, 2014). Therefore, SIM was used as a super-resolation microscopy method to deter- mine the fate of NLRP3 and the inflammasomes. Staining of endoge- nous NLRP3 in LPS/ATP-stimulated microglia revealed several distinct, highly fluorescent NLRP3 puncta/specs in addition to a rather diffuse cytoplasmic staining, which was also observed in non treated cells. The specificity of the NLRP3 staining was confirmed using SIM by co-staining with ASC: An intimate contact between NLRP3 and ASC in activated cells could be observed. Structurally, this interaction resembled the NLRP3-ASC interaction observed in the Salmonella-induced inflammasome (Man et al, 2014), where ASC is forming a ring-like scaffold for NLRP3. One apparent dif- ference is the presence of two or more intervened ASC rings in the inflammasome of LSP/ATP-stimulated microglia, which is not seen in Salmonella-induced inflammasome activation.
circulatory system and the extracellular space of the brain. It is formed by a monolayer of capillary endothelial cells sealed by tight junctions, surrounded by the basal lamina and astrocytic perivascular projections or end-feet. Astrocytes also provide a cellular link to neurons. (B) The BBB regulates the passage of molecules into the brain, lipid soluble molecules can permeate into the brain (transcellular lipophilic pathway), proteins such as transferrin (Tf) are actively endocytosed after binding to specific receptors (receptor-mediated transcytosis), and the uptake of some basic proteins is initiated by a charge interaction with the negatively charged BBB surface (adsorptive-mediated transcytosis). Drug delivery across the BBB may be possible by hijacking these pathways, e.g. using antibodies and nanobodies against transcytosis receptors (e.g. MEM-189, a Fab fragment specific for the Tf receptor (TfR) and FC5, a nanobody that binds α-sialoglycoprotein receptors). Protein cationization might increase uptake by adsorptive transcytosis. (C) Comparison between two routes of administration targeting the brain. The intracerebroventricular (icv) route bypasses the BBB, molecules are infused directly into the cerebrospinal fluid (CSF) that circulates from the ventricles to the brain parenchyma. In contrast, intravenously injected molecules encounter the BBB that restrict their passage into the brain parenchyma. Adapted from Astrocyte- endothelial interactions at the blood-brain barrier by (Abbott et al., 2006).
The developing brain reacts differently from the mature brain when exposed to potentially damaging environment factors or insults. 3-8 Short- and long-term deleterious effects resulting from an interference with normal brain development differ in their extent and quality depending on the nature, the timing, and the extent of the insult. 9 In the past decades, studies in rodents have provided substantial information on brain development. 9-14 Although there are variations in the rates of brain growth among mammals, a comparison of brain development between species is possible. 14,15 The developmental ages of human and rat embryos or fetuses are comparable when anatomical features and histological landmarks are similar in appearance in the two species, even though their exact chronological ages are different. 14 In the CNS, structures are created by cell proliferation, migration, and differentiation as well as the establishment of intercellular connections, i.e. the formation of networks. 9 Normal function requires a specific number of cells with the proper characteristics in the correct location at a specific time. 9 The cycle of neurogenesis of individual neuronal populations has been determined through autoradiography in rodent brain, and extrapolations have been made to the human brain. 9,14 These studies clearly indicate that different brain areas develop at different times during gestation, and within a single brain region, subpopulations of neurons develop at different rates and times. Cerebellar Purkinje cells, for example, develop early (embryonic days 13–15 in the rat, corresponding to gestational weeks 5–7 in humans), whereas granule cells are generated much later (postnatal days 4–19 in the rat, corresponding to gestational weeks 24–40 in humans). 14 Many agents, such as irradiation by x-rays, cause brain damage by interference with cell proliferation, and if the insult occurs during the stage of formation of a certain neuronal subpopulation, the involved cells may not develop.
Translational research depends on identi ﬁcation of patho- mechanisms in vivo and precise follow-up of disease progres- sion. The ﬁrst is important because the complexity of an organism cannot be simulated in cell culture, but is required for develop- ment of therapeutic strategies. The latter is essential to de ﬁne time points for intervention and to precisely monitor possible bene ﬁcial effects. Some animal models, combined with re ﬁnement of clinical imaging tools that are commonly used for AMD patients, enable analysis of cellular and molecular events non-invasively and in real-time. These models reduce signi ﬁcantly the amount of ani- mals needed for experimentation and increase the reliability of the data in terms of translational medicine ( Contag and Bachmann, 2002; Edinger et al., 2002; Rudin and Weissleder, 2003 ). In order to de ﬁne a strategy to inhibit microglia activa- tion as therapeutic target, the accurate knowledge on time- dependence macrophage activation, their spatial distribution and their phenotypes in vivo is needed. This kind of data can be ob- tained by using transgenic mice expressing a microglia/ macrophage-speci ﬁc ﬂuorescence reporter. MacGreen mice spe- ci ﬁcally expressing EGFP in macrophages, mouse models with injected GFP-positive bone marrow cells (chimeric model) or the CX3CR1 GFP/ þ knock-in mice, in which GFP expression is under control of the fractalkine receptor CX3CR1, are the most commonly used animal models for this purpose ( Eter et al., 2008; Joly et al., 2009; Muther et al., 2010; Sasmono et al., 2003 ). In these mouse models, microglia can be visualized by their GFP ﬂuorescence using scanning laser ophthalmoscopy (SLO). The active microglia can be roughly identi ﬁed by their change in cell morphology from rami ﬁed surveying phenotype to the amoeboid invading one. However, the resolution must be high enough to permit a clear and valid identi ﬁcation and separation between active and surveying microglia. Using a confocal SLO, Alt and colleagues ( Alt et al., 2014 ) obtained images which provide the required resolution to safely identify activated microglia and could even establish automated counting of the activated cells. When using conventional SLO, which is also used in the clinic to permit an easy translation of the data from animal model to patients, the raw output doesn't have a high enough resolution for a valid discrimination between surveying and active cells. For example Eter and colleagues ( Eter et al., 2008 ) could visualize the microglia by conventional SLO, but were relying on FACS analysis for their quanti ﬁcation.
24 ]. As early as 1992, Lesnik et al. reported that a tra- cheostomy performed within the first 4 days of injury facilitated weaning from the ventilator in patients who had sustained blunt, multiple organ trauma [ 1 ]. Two years later, D’Amelio et al. identified patients with TBI and multiple trauma to have a shorter duration of mechanical ventilation, as along with decreased ICU and hospital LOS if the tracheostomy was per- formed within 7 days of the injury [ 2 ]. Kluger et al. suggested reduced rates of septic complications when tracheostomy was performed within 3 days of the in- jury in trauma patients [ 3 ]. More recent studies exam- ining similar patient populations reported a reduced LOS in patients undergoing early tracheostomy [ 4 , 5 ]. In 2012, Wang et al. demonstrated that early tra- cheostomy performed by day 10 after severe head in- jury may contribute to a shorter duration of ICU LOS, a lower incidence of Gram-negative microorganism- related nosocomial pneumonia, and a shorter dura- tion of antibiotic use [ 25 ]. Similarly, Rizk et al. re- ported that early tracheostomy resulted in a better overall clinical outcome in TBI patients with associ- ated injuries in at least one other body region, espe- cially when performed in those patients with a reason- able chance of survival [ 24 ]. Recently, Alali et al. re- ported that early tracheostomy within 8 days of injury was associated with a shorter duration of mechanical ventilation and reduced ICU and hospital LOS, but did not reduce hospital mortality [ 26 ].
Glioblastoma (also called glioblastoma multiforme, GBM or grade IV Astrocytoma) is the most common and malignant tumor among glia neoplasms (Louis D.N. et al 2007). GBM is composed by a heterogeneous mixture of scarcely differentiated astrocytes and it usually localizes in the cerebrum, less frequently in the brain stem or in the spinal cord. It affects mostly but not only adults. Unlike other cancers and as all glial tumors, it does not expand out of the central nervous system by spreading through blood, but it invades the brain and spinal cord by active cell migration. GBM usually develops de novo (‘primary’ tumor) but can also arise from a fibrillary (or diffuse, grade II) astrocytoma or from an anaplastic astrocytoma (grade III), in that cases is called ‘secondary’ tumor (Ohgaki H. et al 2007). GBM current standard of care for newly diagnosed patients, established in 2005, consist of maximal surgical resection, followed by temozolomide (TMZ)-based chemotherapy in combination with radiation therapy (RT) (Stupp R. et al 2005). However, due to the high recurrent rates (~90%), patient average survival does not exceed 15 months after diagnosis (Johnson D.R. et al 2012).
The expression levels of osteopontin within glioma cells directly correlate with cell invasiveness and tumor growth. Stably-transformed U87MG glioma cells were transformed with specific small hairpin RNA to knock down osteopontin expression, showed a diminished tumor expansion.  Glioma derived osteopontin is also associated with the extent of neutrophil and macrophage infiltration within the tumor . Regarding its different splice variants in the context of glioma, OPNb seemed to be partially able to compensate for the absence of OPNa and OPNc. While the abrogation of the latter two reduced clonogenic survival, migration as well as proliferation and apoptosis remained unaffected. Only the knockdown of all splice variants leads to a reduction in proliferation and migration and to an increased rate of apoptosis in glioma cells . Recently it has been shown that non-transformed fibroblast conditioned media transfected with Spp1 increased microglia phagocytosis as well as active signal transducer and activator of transcription 1 (STAT1), STAT3 and STAT5. Above that, it leads to an increase in gene expression of Arg1, Smad7, Mmp-14, as well as iNos and Irf7. Further, it was shown that the integrin binding RGD-site of osteopontin is crucial to induce these changes in microglial activation. While osteopontin derived from non-transformed cells primarily leads to a proinflammatory microglial phenotype, glioma cells use MMP and thrombin cleavage to create a short N-terminal osteopontin fragment that induces M2- reprogramming in microglia. A knockdown of osteopontin in a C6 rat model reduced intracranial glioma growth and prevented amoeboid transformation of myeloid cells. It also reduced M2 reprogramming of GAMs .
After 48 h of activation, cells were detached with ice-cold 2.5 mM EDTA in PBS, washed once with PBS, pelleted and lysed in Trizol (Sigma-Aldrich, St. Louis, MO). RNA was isolated using Direct- zol RNA MiniPrep column system (Zymo Research, Irvine, CA) including a DNase I digestion step (15 min, RT). The RNA quantity was measured by NanoDrop (Implen). In addition, 10% of randomly selected RNA samples were tested in the Agilent TapeStation (Agilent Technologies) to check RNA integrity number (RIN) (all samples RIN > 8.5) and to crosscheck the concentration with those measured by NanoDrop. The cDNA synthesis was performed ac- cording to the manufacturer's recommendations using 200 ng RNA for DH82, U937 and human MDMs and 100 ng for canine MDMs using iScript cDNA Synthesis Kit (BioRad, Hercules, CA). For each donor, RNA from all conditions was pooled and used for the reverse transcriptase negative control (RT-). RT-qPCR was performed on a QuantStudio ™12K Flex system (Applied Biosystems, 45 cycles; Tm 59 e61 C; 96 well plate and plate cover (Applied Biosystems) using a Solis Biodyne Supermix (Solis Biodyne, Tartu, Estonia). Target genes were selected according to human literature, concerning a strong discrimination between M1 and M2a macrophages ( Martinez et al., 2006; R€oszer, 2015 ). The primers ( Supplementary Table S1 ) were either selected based on the recent literature or designed using the NCBI Blast and the Oligo Analyzer Tool ( Thornton and Basu, 2011 ). Every plate included pooled RT-per donor and NTC per gene controls. M0 for DH82 and U937 M1 and M2a, GM-CSF for M1 MDMs and M-CSF for M2a MDMs were used as controls. A standard curve was generated and ampli ﬁcation ef- ﬁciency (E) was calculated from the slope s of this curve (E ¼ 10 ( 1/ s) 1) using the QuantStudio 12k Flex Software v1.2.3 (Applied Biosystems). Quanti ﬁcation cycle (Cq) values were corrected by the term Cq log 10 (E þ 1)/log 10 (2) ( Robledo et al., 2014 ). For evalu- ating reference genes in canine macrophages, we tested ten can- didates and chose ornithine decarboxylase antizyme (OAZ1) based on a stable expression pro ﬁle over all activation conditions (data not shown). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference gene for human cells based on Martinez et al. (2006) . After normalizing the data with the reference gene (OAZ1 for DH82 and canine MDMs; GAPDH for human MDMs and U937).
Damit stehe ich nicht alleine. In einer soziologischen Studie über Brain Drain antworteten auf die Frage, wel- che Befürchtungen andere Studierende aus dem ehemali- gen Jugoslawien, die im Ausland leben, vor der Rückkehr haben, 53 Prozent, sie hätten Angst vor »neuen Gesell- schaftsregeln«, 40 Prozent fürchteten, dass ein neuer Krieg ausbrechen werde, 16 Prozent hatten Angst vor der Mafia, 15 Prozent vor Hunger und Armut, weitere 15 Prozent vor der Nicht-Akzeptanz in der Gesellschaft, nach dem sie das Land einmal verlassen hatten. 21 Pro- zent, immerhin ein Fünftel der klugen Köpfchen, sagten, sie würden gar nicht an eine Rückkehr denken.