Sunscreens can preserve human skin microbiome upon erythemal UV exposure.

OBJECTIVE: Ultraviolet radiation (UVR) is a known environmental key factor for premature skin ageing. Only few scientific evidence is available to support the effects of UVR on the skin microbiome. This in vivo pilot study aimed to evaluate the impact on the skin microbiome upon erythemal UV exposure and the protection of UV-exposed skin microbiome by UV filters. METHODS: Ten female volunteers were treated with an sun protection factor (SPF) 20 sunscreen and placebo formulation (without UV filters) on their upper middle backs and irradiated with an erythemal dose (2 MED) by a solar simulator. Skin swabbing samples from four zones (i.e., unexposed, exposed, sunscreen- and placebo-treated on exposed skin) were collected for the microbiome analysis before and 2 h after UV exposure, respectively, and processed via shallow 16S rRNA Amplicon and Shotgun metagenomic sequencing. An in vitro UV method was developed to confirm the protection of isolated bacterial strains by single UV filters and combinations. RESULTS: Alpha diversity was impacted by significant inter-individual differences and by treatment rather than by irradiation. Cutibacterium acnes was found to be the most abundant and a confounding factor for diversity. On a species level, Lactobacillus crispatus was negatively associated with UVR and placebo treatment, whereas there was a positive association with sunscreen treatment. The sunscreen treatment also favoured an interaction network with central Micrococcus genus. The in vitro results showed that both single UV filters and combinations had specific effects on the survival rates of L. crispatus, C. acnes, and Staphylococcus epidermidis. CONCLUSION: We identified potential microorganisms and bacterial interactions that were associated with an SPF 20 sunscreen treatment. The specific protection of L. crispatus as a key player in the UV-exposed skin microbiome and reduction of C. acnes population by UV filters might lead to new cosmetic concepts for photoprotection.

OBJECTIF: Le rayonnement ultraviolet (RUV) est un facteur environnemental clé connu du vieillissement prématuré de la peau. Peu de preuves scientifiques sont disponibles pour étayer les effets des RUV sur le microbiome cutané. Cette étude pilote in vivo visait à évaluer l'impact sur le microbiome cutané d'une exposition érythèmateuse aux UV et la protection du microbiome cutané exposé aux UV par des filtres UV. MÉTHODES: Dix volontaires de sexe féminin ont été traitées avec une crème solaire SPF 20 et une formulation placebo (sans filtres UV) sur la partie supérieure du centre du dos et irradiées avec une dose érythémateuse (2 MED) par un simulateur solaire. Des échantillons de peau prélevés par écouvillonnage dans quatre zones (c.-à-d., zone non exposée, zone exposée, zone traitée avec un écran solaire et zone traitée avec un placebo sur la peau exposée) ont été prélevés pour l'analyse du microbiome avant et 2 heures après l'exposition aux UV, respectivement, et traités par séquençage superficiel d'amplicon de l'ARN 16S et métagénomique shotgun. Une méthode UV in vitro a été développée pour confirmer la protection des souches bactériennes isolées par des filtres UV individuels et des combinaisons de filtres. RÉSULTATS: La diversité alpha a été affectée par des différences interindividuelles significatives et par le traitement plutôt que par l'irradiation. Le cutibacterium acnes s'est avéré être le facteur le plus abondant et confondant pour la diversité. Au niveau de l'espèce, le Lactobacillus crispatus était négativement associé au traitement par RUV et placebo, tandis qu'on observait une association positive avec le traitement par écran solaire. Le traitement par crème solaire favorisait également un réseau d'interactions avec le genre Micrococcus central. Les résultats in vitro ont montré que les filtres UV individuels et les associations de filtres avaient des effets spécifiques sur les taux de survie de L. crispatus, C. acnes, et S. epidermidis. CONCLUSION: Nous avons identifié des micro-organismes et des interactions bactériennes potentiels qui étaient associés à un traitement par crème solaire SPF 20. La protection spécifique de L. crispatus en tant qu'acteur clé dans le microbiome cutané exposé aux UV et la réduction de la population de C. acnes par des filtres UV pourraient conduire à de nouveaux concepts cosmétiques de photoprotection.

5-α reductase inhibition by Epilobioum fleischeri extract modulates facial microbiota structure.

BACKGROUND: Facial skin is a particularly complex environment made of different skin types such as sebaceous (forehead) and dry (cheeks). The skin microbiota composition on different facial sites has not yet been addressed. METHODS: We conducted a 4-week-long, single-centre, randomized and placebo-controlled clinical study involving 23 Caucasian females. We assessed both bacterial composition on five different facial areas and the microbiome modulatory effects resulting from the topical application of a plant extract (Epilobium fleischeri). Skin microbiome samples were collected before and after 4 weeks of product application. Microbiota profiling was performed via 16S rRNA gene sequencing, and relative abundance data were used to calculate differentials via a multinomial regression model. RESULTS: Via 'reference frames', we observed shifts in microbial composition after 4 weeks of twice-daily product application and identify certain microbiota species, which were positively associated with the application of the product containing the Epilobium fleischeri extract. Staphylococcus hominis, Staphylococcus epidermidis, and Micrococcus yunnanensis appeared to be significantly enriched in the final microbiota composition of the active treatment group. CONCLUSION: Facial skin was found to be colonized by an heterogenous microbiota, and the Epilobium fleischeri extract had a modulatory effect on commensal bacteria on the different facial sites.

CONTEXTE: la peau du visage est un environnement particulièrement complexe où l'on trouve des peaux de plusieurs types, par exemple grasse (sur le front) et sèche (sur les joues). La composition du microbiote cutané sur différentes zones du visage n'a pas encore été abordée. MÉTHODES: nous avons mené une étude clinique de 4 semaines monocentrique, randomisée et contrôlée par placebo sur 23 femmes de type caucasien. Nous avons évalué à la fois la composition bactérienne sur cinq zones différentes du visage et les effets modulateurs du microbiome résultant de l'application topique d'un extrait de plante (Epilobium fleischeri). Des échantillons de microbiome cutané ont été prélevés avant et après 4 semaines d'application du produit. Un profilage du microbiote a été mené par séquençage du gène de l'ARNr 16S, des données d'abondance relative ont été utilisées pour calculer les différentiels via un modèle de régression multinomiale. RÉSULTATS: nos cadres de référence nous ont permis d'observer des changements de composition microbienne après 4 semaines d'application deux fois par jour du produit et nous avons identifié certaines espèces de microbiote qui ont été positivement associées à l'application du produit contenant l'extrait d'Epilobium fleischeri. Les taux de Staphylococcus hominis, Staphylococcus epidermidis et Micrococcus yunnanensis semblaient significativement plus élevés dans la composition finale du microbiote du groupe de traitement actif. CONCLUSION: la peau du visage s'est avérée colonisée par un microbiote hétérogène, et l'extrait d'Epilobium fleischeri a eu un effet modulateur sur les bactéries commensales des différentes zones du visage.

Cold non-ischemic heart preservation with continuous perfusion prevents early graft failure in orthotopic pig-to-baboon xenotransplantation.

BACKGROUND: Successful preclinical transplantations of porcine hearts into baboon recipients are required before commencing clinical trials. Despite years of research, over half of the orthotopic cardiac xenografts were lost during the first 48 hours after transplantation, primarily caused by perioperative cardiac xenograft dysfunction (PCXD). To decrease the rate of PCXD, we adopted a preservation technique of cold non-ischemic perfusion for our ongoing pig-to-baboon cardiac xenotransplantation project. METHODS: Fourteen orthotopic cardiac xenotransplantation experiments were carried out with genetically modified juvenile pigs (GGTA1- KO/hCD46/hTBM) as donors and captive-bred baboons as recipients. Organ preservation was compared according to the two techniques applied: cold static ischemic cardioplegia (IC; n = 5) and cold non-ischemic continuous perfusion (CP; n = 9) with an oxygenated albumin-containing hyperoncotic cardioplegic solution containing nutrients, erythrocytes and hormones. Prior to surgery, we measured serum levels of preformed anti-non-Gal-antibodies. During surgery, hemodynamic parameters were monitored with transpulmonary thermodilution. Central venous blood gas analyses were taken at regular intervals to estimate oxygen extraction, as well as lactate production. After surgery, we measured troponine T and serum parameters of the recipient's kidney, liver and coagulation functions. RESULTS: In porcine grafts preserved with IC, we found significantly depressed systolic cardiac function after transplantation which did not recover despite increasing inotropic support. Postoperative oxygen extraction and lactate production were significantly increased. Troponin T, creatinine, aspartate aminotransferase levels were pathologically high, whereas prothrombin ratios were abnormally low. In three of five IC experiments, PCXD developed within 24 hours. By contrast, all nine hearts preserved with CP retained fully preserved systolic function, none showed any signs of PCXD. Oxygen extraction was within normal ranges; serum lactate as well as parameters of organ functions were only mildly elevated. Preformed anti-non-Gal-antibodies were similar in recipients receiving grafts from either IC or CP preservation. CONCLUSIONS: While standard ischemic cardioplegia solutions have been used with great success in human allotransplantation over many years, our data indicate that they are insufficient for preservation of porcine hearts transplanted into baboons: Ischemic storage caused severe impairment of cardiac function and decreased tissue oxygen supply, leading to multi-organ failure in more than half of the xenotransplantation experiments. In contrast, cold non-ischemic heart preservation with continuous perfusion reliably prevented early graft failure. Consistent survival in the perioperative phase is a prerequisite for preclinical long-term results after cardiac xenotransplantation.

Microbial Reference Frames Reveal Distinct Shifts in the Skin Microbiota after Cleansing.

Skin cleansing represents a process of mechanical and chemical removal of dirt, pollutants as well as microbiota from the skin. While skin cleansing can help maintain good health, protect us from infections, illnesses and ailments, skin cleansing can also strip away lipids and moisture from the skin, leading to irritation, barrier impairment and disturbance of the delicate cutaneous microbiome. This study investigated how skin cleansing impacts skin's microbial composition. Thirty Caucasian women were enrolled in a placebo controlled clinical study where participants applied on their volar forearms a liquid body wash twice daily for 1 week in order to mimic frequent showering. Skin microbiome samples were collected by swabbing at defined timepoints and 16S rRNA sequencing was performed. Using "reference frames", we could identify shifts in the microbial composition and several microbiota were identified as being characteristically associated with the presence of saccharide isomerate, a well-known skin moisturizer. The microbial shift was quite immediate, and we could observe it already at 1 h post cleansing. Interestingly, the new microbial composition reached a certain dynamic equilibrium at day 1 which was then maintained until the end of the study. Paracoccus marcusii, a potentially beneficial carotenoid-producer microorganism, was enriched by the active treatment and, at the same time, the abundance of several potential pathogenic taxa, Brevibacterium casei and Rothia mucilaginosa, diminished.

Pig-to-non-human primate heart transplantation: The final step toward clinical xenotransplantation?

BACKGROUND: The demand for donated human hearts far exceeds the number available. Xenotransplantation of genetically modified porcine organs provides an alternative. In 2000, an Advisory Board of the International Society for Heart and Lung Transplantation set the benchmark for commencing clinical cardiac xenotransplantation as consistent 60% survival of non-human primates after life-supporting porcine heart transplantations. Recently, we reported the stepwise optimization of pig-to-baboon orthotopic cardiac xenotransplantation finally resulting in consistent success, with 4 recipients surviving 90 (n = 2), 182, and 195 days. Here, we report on 4 additional recipients, supporting the efficacy of our procedure. RESULTS: The first 2 additional recipients succumbed to porcine cytomegalovirus (PCMV) infections on Days 15 and 27, respectively. In 2 further experiments, PCMV infections were successfully avoided, and 3-months survival was achieved. Throughout all the long-term experiments, heart, liver, and renal functions remained within normal ranges. Post-mortem cardiac diameters were slightly increased when compared with that at the time of transplantation but with no detrimental effect. There were no signs of thrombotic microangiopathy. The current regimen enabled the prolonged survival and function of orthotopic cardiac xenografts in altogether 6 of 8 baboons, of which 4 were now added. These results exceed the threshold set by the Advisory Board of the International Society for Heart and Lung Transplantation. CONCLUSIONS: The results of our current and previous experimental cardiac xenotransplantations together fulfill for the first time the pre-clinical efficacy suggestions. PCMV-positive donor animals must be avoided.

Genetically encoded Ca2+ -sensor reveals details of porcine endothelial cell activation upon contact with human serum.

The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca2+ -messaging during the activation of endothelial cells is well recognized and routinely measured by synthetic Ca2+ -sensitive fluorophors. However, these compounds require fresh loading immediately before each experiment and in particular when grown in state-of-the-art 3D cell culture systems, endothelial cells are difficult to access with such sensors. Therefore, we developed transgenic pigs expressing a Ca2+ -sensitive protein and examined its principal characteristics. Primary transgenic endothelial cells stimulated by ATP showed a definite and short influx of Ca2+ into the cytosol, whereas exposure to human serum resulted in a more intense and sustained response. Surprisingly, not all endothelial cells reacted identically to a stimulus, rather activation took place in adjacent cells in a timely decelerated way and with distinct intensities. This effect was again more pronounced when cells were stimulated with human serum. Finally, we show clear evidence that antibody binding alone significantly activated endothelial cells, whereas antibody depletion dramatically reduced the stimulatory potential of serum. Transgenic porcine endothelial cells expressing a Ca2+ -sensor represent an interesting tool to dissect factors inducing activation of porcine endothelial cells after exposure to human blood or serum.

3D Cell-Culture Models for the Assessment of Anticoagulant and Anti-Inflammatory Properties of Endothelial Cells.

Endothelial cells (EC) play a crucial role in the pathophysiology of cardiovascular diseases, ischemia/reperfusion injury, and graft rejection in (xeno-)transplantation. In such nonphysiological conditions, EC are known to lose their quiescent phenotype and switch into an actively pro-inflammatory, procoagulant, and anti-fibrinolytic state. This case happens essentially because the endothelial glycocalyx-a layer of proteoglycans and glycoproteins covering the luminal surface of the endothelium-is shed. Heparan sulfate, one of the main components of the endothelial glycocalyx, contributes to its negative charge. In addition, many plasma proteins such as antithrombin III, superoxide dismutase, C1 inhibitor, and growth factors and cytokines bind to heparan sulfate and by this scenario contribute to the establishment of an anticoagulant and anti-inflammatory endothelial surface. Shedding of the glycocalyx results in a loss of plasma proteins from the endothelial surface, and this phenomenon causes the switch in phenotype. Particularly in xenotransplantation, both hyperacute and acute vascular rejection are characterized by coagulation dysregulation, a situation in which EC are the main players.Since many years, EC have been used in vitro in 2D flatbed cell culture models, with or without the application of shear stress. Such models have also been used to assess the effect of human transgenes on complement- and coagulation-mediated damage of porcine EC in the context of xenotransplantation. The methods described in this chapter include the analysis of endothelial cell-blood interactions without the necessity of using anticoagulants as the increased EC surface-to-volume ratio allows for natural anticoagulation of blood. Furthermore, this chapter contains the description of a novel microfluidic in vitro model carrying important features of small blood vessels, such as a 3D round-section geometry, shear stress, and pulsatile flow-all this in a closed circuit, recirculating system aiming at reproducing closely the in vivo situation in small vessels.

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

BACKGROUND: Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. METHODS: Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. RESULTS: Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. CONCLUSION: We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

Author Correction: Consistent success in life-supporting porcine cardiac xenotransplantation.

In this Letter, Mayuko Kurome and Valeri Zakhartchenko have been added to the author list (affiliated with Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany). The author list and 'Author contributions' section have been corrected online; see accompanying Amendment.

Consistent success in life-supporting porcine cardiac xenotransplantation.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1-3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation7.

3D artificial round section micro-vessels to investigate endothelial cells under physiological flow conditions.

In the context of xenotransplantation, in ischemia/reperfusion injury as well as in cardiovascular research, the study of the fascinating interplay between endothelial cells (EC) and the plasma cascade systems often requires in vitro models. Blood vessels are hardly reproducible with standard flat-bed culture systems and flow-plate assays are limited in their low surface-to-volume ratio which impedes the study of the anticoagulant properties of the endothelial cells. According to the 3R regulations (reduce, replace and refine animal experimentation) we developed a closed circuit microfluidic in vitro system in which endothelial cells are cultured in 3D round section microchannels and subjected to physiological, pulsatile flow. In this study, a 3D monolayer of porcine aortic EC was perfused with human serum to mimic a xenotransplantation setting. Complement as well as EC activation was assessed in the presence or absence of complement inhibitors showing the versatility of the model for drug testing. Complement activation products as well as E-selectin expression were detected and visualized in situ by high resolution confocal microscopy. Furthermore, porcine pro-inflammatory cytokines as well as soluble complement components in the recirculating fluid phase were detected after human serum perfusion providing a better overview of the artificial vascular environment.

Multiple genetically modified GTKO/hCD46/HLA-E/hß2-mg porcine hearts are protected from complement activation and natural killer cell infiltration during ex vivo perfusion with human blood.

BACKGROUND: In pig-to-human xenotransplantation, early cellular rejection reactions are mediated by natural killer cells (NK cells). Human NK cells are inhibited by HLA-E via CD94/NKG2A receptors. To protect porcine grafts against human NK cell responses, transgenic GTKO pigs expressing hCD46 and HLA-E have been generated. The aim of this study was to test the effect of this genetic modification on xenogeneic, and in particular human NK cell response, using an ex vivo perfusion model of pig hearts with human blood. METHODS: Cardiopleged and explanted genetically modified (gm) pig hearts (GTKO/hCD46/HLA-E/hß2-microglobulin) and wild-type (wt) controls (n = 6 each) were reperfused and tested in an 8 hours ex vivo perfusion system using freshly drawn human blood. Cardiac function was evaluated during a 165-minute period in working heart mode. Myocardial damage, antibody deposition, complement activation, and coagulation parameters were evaluated histologically at the end of perfusion. The number of NK cells in the perfusate was determined by flow cytometry at baseline and at 8 hours; tissue infiltration by NK cells was quantified by immunofluorescence microscopy using NKp46 staining of frozen sections. RESULTS: Deposition of IgG (1.2 ± 1 × 107 vs 8.8 ± 2.9 × 106 ; P < .01), IgM (4.4 ± 3.7 × 106 vs 1.7 ± 1.2 × 106 ; P < .01), and the complement activation product C4b/c (3.5 ± 1.3 × 106 vs 2.3 × 106  ± 9.4 × 105 ; P > .01) was lower in gm than wt hearts. NK cell percentages of leukocytes in the perfusate decreased from 0.94 ± 0.77% to 0.21 ± 0.25% (P = .04) during xenoperfusion of wt hearts. In contrast, the ratio of NK cells did not decrease significantly in the gm hearts. In this group, NK cell myocardial infiltration after 480 minutes of perfusion was lower than in wt organs (2.5 ± 3.7 × 104 /mm3 vs 1.3 ± 1.4 × 105 /mm3 ; P = .0001). The function of gm hearts was better preserved compared to wt organs, as demonstrated by higher cardiac index during the first 2 hours of ex vivo perfusion. CONCLUSION: GTKO, hCD46, and HLA-E expression in porcine hearts reduced complement deposition, complement dependent injury, and myocardial NK cell infiltration during perfusion with human blood. This tested combination of genetic modifications may minimize damage from acute human-anti-pig rejection reactions and improve myocardial function after xenotransplantation.

Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA-E/human CD46 double-transgenic pig limbs with human blood.

BACKGROUND: In pig-to-human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non-classical human leukocyte antigen-E (HLA-E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood. METHODS: Six pig forelimbs per group, respectively, stemming from either wild-type (wt) or HLA-E/hCD46 double-transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12 hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells. RESULTS: Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60 minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10 minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12 hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA-E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells. CONCLUSIONS: Ex vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell-mediated rejection mechanisms of vascularized pig-to-human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA-E/hCD46 in pig limbs provides partial protection from human NK cell-mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood.

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the "natural" anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

Fluorescence-based gene reporter plasmid to track canonical Wnt signaling in ENS inflammation.

In several gut inflammatory or cancer diseases, cell-cell interactions are compromised, and an increased cytoplasmic expression of ß-catenin is observed. Over the last decade, numerous studies provided compelling experimental evidence that the loss of cadherin-mediated cell adhesion can promote ß-catenin release and signaling without any specific activation of the canonical Wnt pathway. In the present work, we took advantage of the ability of lipofectamine-like reagent to cause a synchronous dissociation of adherent junctions in cells isolated from the rat enteric nervous system (ENS) for obtaining an in vitro model of deregulated ß-catenin signaling. Under these experimental conditions, a green fluorescent protein Wnt reporter plasmid called ΔTop_EGFP3a was successfully tested to screen ß-catenin stabilization at resting and primed conditions with exogenous Wnt3a or lipopolysaccharide (LPS). ΔTop_EGFP3a provided a reliable and strong fluorescent signal that was easily measurable and at the same time highly sensitive to modulations of Wnt signaling following Wnt3a and LPS stimulation. The reporter gene was useful to demonstrate that Wnt3a exerts a protective activity in the ENS from overstimulated Wnt signaling by promoting a downregulation of the total ß-catenin level. Based on this evidence, the use of ΔTop_EGFP3a reporter plasmid could represent a more reliable tool for the investigation of Wnt and cross-talking pathways in ENS inflammation.