Peripheral Lymph Node Addressins are Expressed on Skin Endothelial Cells

The term “peripheral node addressins” describes a set of several endothelial adhesion molecules, which collectively bind to L-selectin and react with monoclonal antibody MECA-79. They regulate lymphocyte recirculation through peripheral nodes. Their expression is thought to be restricted to a specialized vascular segment within the node, called the high endothelial venule. In certain chronic skin diseases, however, postcapillary venules of the skin may also acquire a high endothelial venule-like morphology. Employing immunohistochemistry on cryostat sections, we found these skin endothelial cells – like peripheral node high endothelial venules – to be reactive with monoclonal antibody MECA-79. Tissue lysates from the same specimens were then analyzed by immunoprecipitation using recombinant human L-selectin Fc-chimeras followed by immunoblotting using monoclonal antibody MECA-79. In contrast to peripheral node endothelium, which mainly expressed peripheral node addressin moieties of molecular sizes 90–110 kDa and 160 kDa, endothelial cells in cutaneous T cell lymphoma skin lesions expressed an additional and not yet defined 220 kDa peripheral node addressin-like molecule. Most surprisingly, even in normal skin specimens, we found a distinct subset of endothelial cells located around hair follicles constitutively expressing 90–110 kDa peripheral node addressin-like moieties. It is intriguing to speculate that – in analogy to the role of peripheral node addressins in peripheral nodes – the induced expression of peripheral node addressins in chronic T cell mediated skin diseases is responsible for a sustained lymphocyte recruitment. The constitutive expression of peripheral node addressins on perifollicular endothelium may serve for a continuous lymphocyte recirculation through normal skin

Peptide Bbeta(15-42) preserves endothelial barrier function in shock

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn -/- mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock

Combination of Dacarbazine and Dimethylfumarate Efficiently Reduces Melanoma Lymph Node Metastasis

Dimethylfumarate (DMF) has been shown to reduce melanoma growth and metastasis in animal models. We addressed the question of whether DMF is as effective in its antitumor activity as the US Food and Drug Administration–approved alkylating agent dacarbazine (DTIC). We also tested the possibility of an improved antitumoral effect when both therapeutics were used together. Using our severe combined immunodeficiency (SCID) mouse model, in which xenografted human melanoma cells metastasize from primary skin sites to sentinel nodes, we show that these treatments, alone or in combination, reduce tumor growth at primary sites. Our main finding was that metastasis to sentinel nodes is significantly delayed only in mice treated with a combination of DTIC and DMF. Subsequent experiments were able to show that a combination of DTIC/DMF significantly reduced lymph vessel density in primary tumors as examined by real-time PCR and immunohistochemistry. In addition, DTIC/DMF treatment significantly impaired melanoma cell migration in vitro. In vivo, DTIC/DMF therapy significantly reduced mRNA expression and protein concentration of the promigratory chemokines CXCL2 and CXCL11. In addition, our data suggest that this xenotransplantation model is suitable for preclinical testing of various combinations of antimelanoma agents

UV spectrophotometry method for the monitoring of galacto-oligosaccharides production

Monitoring the industrial production of galacto-oligosaccharides (GOS) requires a fast and accurate methodology able to quantify, in real time, the substrate level and the product yield. In this work, a simple, fast and inexpensive UV spectrophotometric method, together with partial least squares regression (PLS) and artificial neural networks (ANN), was applied to simultaneously estimate the products (GOS) and the substrate (lactose) concentrations in fermentation samples. The selected multiple models were trained and their prediction abilities evaluated by cross-validation and external validation being the results obtained compared with HPLC measurements. ANN models, generated from absorbance spectra data of the fermentation samples, gave, in general, the best performance being able to accurately and precisely predict lactose and total GOS levels, with standard error of prediction lower than 13 g kg 1 and coefficient of determination for the external validation set of 0.93–0.94, showing residual predictive deviations higher than five, whereas lower precision was obtained with the multiple model generated with PLS. The results obtained show that UV spectrophotometry allowed an accurate and non-destructive determination of sugars in fermentation samples and could be used as a fast alternative method for monitoring GOS production.Fundação para a Ciência e a Tecnologia (FCT) - Bolsa de doutouramento SFRH/BDE/15510/2004Agência da Inovação – Programa IDEIA (Potugal

Differences in biocompatibility of microneedles from cyclic olefin polymers with human endothelial and epithelial skin cells

Microneedles are promising devices for transdermal delivery and diagnostic applications, due to their minimally invasive and painless nature of application. However, so far, applications are limited to small scale research projects. Material selection and production for larger projects remain a challenge. In vitro testing using human cell culture could bridge the gap between cost effective screening of suitable materials and concerns for safety and ethics. In this study, materials were tested for effects on viability and morphology of human endothelial cells and keratinocytes. In addition, materials were assessed for their potential to influence cellular differentiation and barrier formation. Elution-based testing of inflammatory markers revealed no negative effects in all applied tests, whereas the assessment of differentiation markers on cells in direct contact with the material showed differences and allowed the selection of candidate materials for future medical device applications. This study illustrates that elution-based biocompatibility testing can paint an incomplete picture. Advanced staining techniques and cell types specific for the application of the medical device improve material selection to reduce and replace animal testing at an early stage in the development process. © 2018 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 505–512, 2019

Human Skin/SCID Mouse Chimeras as an In Vivo Model for Human Cutaneous Mast Cell Hyperplasia

Human skin xenografted to mice with severe combined immunodeficiency syndrome (SCID) was evaluated to determine the integrity and fate of human dermal mast cells. There was an approximately 3-fold increase in number of dermal mast cells by 3 mo after engraftment (p < 0.05). These cells were responsive to conventional mast cell secretagogues and were confirmed to be of human origin by ultrastructural characterization of granule substructure and by reactivity for the human mast cell proteinase, chymase. CD1a+ Langerhans cells, also bone marrow–derived cells, failed to show evidence of concomitant hyperplasia, and increased mast cell number was not associated with alterations in number of dermal vascular profiles identified immunohistochemically for human CD31. RT-PCR analysis demonstrated human but not murine stem cell factor (SCF; also termed mast cell growth factor, c-kit ligand) mRNA in xenografts. Epidermal reactivity for stem cell factor protein shifted from a cytoplasmic pattern to an intercellular pattern by 3 mo after engraftment, suggesting a secretory phenotype, as previously documented for human cutaneous mastocytosis. The majority (>90%) of mast cells demonstrated membrane reactivity for human SCF at the time points of peak hyperplasia. These data establish SCID mouse recipients of human skin xenografts as a potential in vivo model for cutaneous mast cell hyperplasia

Cell Type-Specific Roles of NF-κB Linking Inflammation and Thrombosis

The transcription factor NF-κB is a central mediator of inflammation with multiple links to thrombotic processes. In this review, we focus on the role of NF-κB signaling in cell types within the vasculature and the circulation that are involved in thrombo-inflammatory processes. All these cells express NF-κB, which mediates important functions in cellular interactions, cell survival and differentiation, as well as expression of cytokines, chemokines, and coagulation factors. Even platelets, as anucleated cells, contain NF-κB family members and their corresponding signaling molecules, which are involved in platelet activation, as well as secondary feedback circuits. The response of endothelial cells to inflammation and NF-κB activation is characterized by the induction of adhesion molecules promoting binding and transmigration of leukocytes, while simultaneously increasing their thrombogenic potential. Paracrine signaling from endothelial cells activates NF-κB in vascular smooth muscle cells and causes a phenotypic switch to a “synthetic” state associated with a decrease in contractile proteins. Monocytes react to inflammatory situations with enforced expression of tissue factor and after differentiation to macrophages with altered polarization. Neutrophils respond with an extension of their life span—and upon full activation they can expel their DNA thereby forming so-called neutrophil extracellular traps (NETs), which exert antibacterial functions, but also induce a strong coagulatory response. This may cause formation of microthrombi that are important for the immobilization of pathogens, a process designated as immunothrombosis. However, deregulation of the complex cellular links between inflammation and thrombosis by unrestrained NET formation or the loss of the endothelial layer due to mechanical rupture or erosion can result in rapid activation and aggregation of platelets and the manifestation of thrombo-inflammatory diseases. Sepsis is an important example of such a disorder caused by a dysregulated host response to infection finally leading to severe coagulopathies. NF-κB is critically involved in these pathophysiological processes as it induces both inflammatory and thrombotic responses

High circulating activin A level is associated with tumor progression and predicts poor prognosis in lung adenocarcinoma

Activin A (ActA)/follistatin (FST) signaling has been shown to be deregulated in different tumor types including lung adenocarcinoma (LADC). Here, we report that serum ActA protein levels are significantly elevated in LADC patients (n=64) as compared to controls (n=46, p=0.015). ActA levels also correlated with more advanced disease stage (p<0.0001) and T (p=0.0035) and N (p=0.0002) factors. M1 patients had significantly higher ActA levels than M0 patients (p<0.001). High serum ActA level was associated with poor overall survival (p<0.0001) and was confirmed as an independent prognostic factor (p=0.004). Serum FST levels were increased only in female LADC patients (vs. female controls, p=0.031). Two out of five LADC cell lines secreted biologically active ActA, while FST was produced in all of them. Transcripts of both type I and II ActA receptors were detected in all five LADC cell lines. In conclusion, our study does not only suggest that measuring blood ActA levels in LADC patients might improve the prediction of prognosis, but also indicates that this parameter might be a novel non-invasive biomarker for identifying LADC patients with organ metastases

Galacto-Oligosaccharides : production, properties, applications, and significance as prebiotics

Galacto-oligosaccharides (GOS) have now been definitely established as prebiotic ingredients after in vitro and animal and human in vivo studies. Currently, GOS are produced by glycoside hydrolases (GH) using lactose as substrate. Converting lactose into GOS by GH results in mixtures containing GOS of different degrees of polymerization (DP), unreacted lactose, and monomeric sugars (glucose and galactose). Recent and future developments in the production of GOS aim at delivering purer and more efficient mixtures. To produce high-GOS-content mixtures, GH should not only have good ability to catalyze the transgalactosylation reaction relative to hydrolysis, but also have low affinity for the GOS formed relative to the affinity for lactose. In this article, several microbial GH, proposed for the synthesis of GOS, are hierarchized according to the referred performance indicators. In addition, strategies for process improvement are discussed. Besides the differences in purity of GOS mixtures, differences in the position of the glycosidic linkages occur, because different enzymes have different regiochemical selectivity. Depending on oligosaccharide composition, GOS products will vary in terms of prebiotic activity, as well as other physiological effects. This review focuses on GOS production from synthesis to purification processes. Physicochemical characteristics, physiological effects, and applications of these prebiotic ingredients are summarized. Regulatory aspects of GOS-containing food products are also highlighted with emphasis on the current process of health claims evaluation in Europe.Agência da Inovação-Progama IDEIA (Portugal)Fundação para a Ciência e a Tecnologia (FCT