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Farber disease (FD) is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments are clinically available and affected patients have a severely shortened lifespan. Due to the low incidence, the pathogenesis of FD is still poorly understood. Here, we report a novel acid ceramidase mutant mouse model that enables the study of pathogenic mechanisms of FD and ceramide accumulation. Asah1(tmEx1) mice were generated by deletion of the acid ceramidase signal peptide sequence. The effects on lysosomal targeting and activity of the enzyme were assessed. Ceramide and sphingomyelin levels were quantified by liquid chromatography tandem-mass spectrometry (LC-MS/MS) and disease manifestations in several organ systems were analyzed by histology and biochemistry. We show that deletion of the signal peptide sequence disrupts lysosomal targeting and enzyme activity, resulting in ceramide and sphingomyelin accumulation. The affected mice fail to thrive and die early. Histiocytic infiltrations were observed in many tissues, as well as lung inflammation, liver fibrosis, muscular disease manifestations and mild kidney injury. Our new mouse model mirrors human FD and thus offers further insights into the pathogenesis of this disease. In the future, it may also facilitate the development of urgently needed therapies.
Ventilator-induced lung injury is aggravated by antibiotic mediated microbiota depletion in mice
(2018)
BackgroundAntibiotic exposure alters the microbiota, which can impact the inflammatory immune responses. Critically ill patients frequently receive antibiotic treatment and are often subjected to mechanical ventilation, which may induce local and systemic inflammatory responses and development of ventilator-induced lung injury (VILI). The aim of this study was to investigate whether disruption of the microbiota by antibiotic therapy prior to mechanical ventilation affects pulmonary inflammatory responses and thereby the development of VILI.MethodsMice underwent 6-8weeks of enteral antibiotic combination treatment until absence of cultivable bacteria in fecal samples was confirmed. Control mice were housed equally throughout this period. VILI was induced 3 days after completing the antibiotic treatment protocol, by high tidal volume (HTV) ventilation (34ml/kg; positive end-expiratory pressure=2 cmH(2)O) for 4h. Differences in lung function, oxygenation index, pulmonary vascular leakage, macroscopic assessment of lung injury, and leukocyte and lymphocyte differentiation were assessed. Control groups of mice ventilated with low tidal volume and non-ventilated mice were analyzed accordingly.ResultsAntibiotic-induced microbiota depletion prior to HTV ventilation led to aggravation of VILI, as shown by increased pulmonary permeability, increased oxygenation index, decreased pulmonary compliance, enhanced macroscopic lung injury, and increased cytokine/chemokine levels in lung homogenates.ConclusionsDepletion of the microbiota by broad-spectrum antibiotics prior to HTV ventilation renders mice more susceptible to developing VILI, which could be clinically relevant for critically ill patients frequently receiving broad-spectrum antibiotics.
Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids found in fish and algae, elicit substantial toxic effects in various human cell lines and have a considerable impact on cellular energy levels. The underlying mode of action, however, is still unknown. The present study analyzes the effects of two AsHCs (AsHC 332 and AsHC 360) on the expression of 44 genes covering DNA repair, stress response, cell death, autophagy, and epigenetics via RT-qPCR in human liver (HepG2) cells. Both AsHCs affected the gene expression, but to different extents. After treatment with AsHC 360, flap structure-specific endonuclease 1 (FEN1) as well as xeroderma pigmentosum group A complementing protein (XPA) and (cytosine-5)-methyltransferase 3A (DNMT3A) showed time- and concentration-dependent alterations in gene expression, thereby indicating an impact on genomic stability. In the subsequent analysis of epigenetic markers, within 72 h, neither AsHC 332 nor AsHC 360 showed an impact on the global DNA methylation level, whereas incubation with AsHC 360 increased the global DNA hydroxymethylation level. Analysis of cell extracts and cell media by HPLC-mass spectrometry revealed that both AsHCs were considerably biotransformed. The identified metabolites include not only the respective thioxo-analogs of the two AsHCs, but also several arsenic-containing fatty acids and fatty alcohols, contributing to our knowledge of biotransformation mechanisms of arsenolipids.
Major depressive disorder (MDD) is a common and severe disease characterized by mood changes, somatic alterations, and often suicide. MDD is treated with antidepressants, but the molecular mechanism of their action is unknown. We found that widely used antidepressants such as amitriptyline and fluoxetine induce autophagy in hippocampal neurons via the slow accumulation of sphingomyelin in lysosomes and Golgi membranes and of ceramide in the endoplasmic reticulum (ER). ER ceramide stimulates phosphatase 2A and thereby the autophagy proteins Ulk, Beclin, Vps34/Phosphatidylinositol 3-kinase, p62, and Lc3B. Although treatment with amitriptyline or fluoxetine requires at least 12 days to achieve sphingomyelin accumulation and the subsequent biochemical and cellular changes, direct inhibition of sphingomyelin synthases with tricyclodecan-9-yl-xanthogenate (D609) results in rapid (within 3 days) accumulation of ceramide in the ER, activation of autophagy, and reversal of biochemical and behavioral signs of stress-induced MDD. Inhibition of Beclin blocks the antidepressive effects of amitriptyline and D609 and induces cellular and behavioral changes typical of MDD. These findings identify sphingolipid-controlled autophagy as an important target for antidepressive treatment methods and provide a rationale for the development of novel antidepressants that act within a few days.
Due to the low cutaneous bioavailability of tacrolimus (TAC), penetration enhancers are used to improve its penetration into the skin. However, poor loading capacity, non-biodegradability, toxicity, and in some cases inefficient skin penetration are challenging issues that hamper their applications for the dermal TAC delivery. Here we present poly(lactide-co-glycerol) (PLG) as a water soluble, biodegradable, and biocompatible TAC-carrier with high loading capacity (14.5% w/w for TAC) and high drug delivery efficiencies into the skin. PLG was synthesized by cationic ring-opening copolymerization of a mixture of glycidol and lactide and showed 35 nm and 300 nm average sizes in aqueous solutions before and after loading of TAC, respectively. Delivery experiments on human skin, quantified by fluorescence microscopy and LC-MS/MS, showed a high ability for PLG to deposit Nile red and TAC into the stratum corneum and viable epidermis of skin in comparison with Protopic (R) (0.03% w/w, TAC ointment). The cutaneous distribution profile of delivered TAC proved that 80%, 16%, and 4% of the cutaneous drug level was deposited in the stratum corneum, viable epidermis, and upper dermis, respectively. TAC delivered by PLG was able to efficiently decrease the IL-2 and TSLP expressions in human skin models. Taking advantage of the excellent physicochemical and biological properties of PLG, it can be used for efficient dermal TAC delivery and potential treatment of inflammatory skin diseases.
Objectives: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. Design: Controlled, in vitro, ex vivo, and in vivo laboratory study. Subjects: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. Interventions: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. Measurements and Main Results: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1(-/-) mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. Conclusions: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.
Chronic psychosocial stress adversely affects human morbidity and is a risk factor for inflammatory disorders, liver diseases, obesity, metabolic syndrome, and major depressive disorder (MDD). In recent studies, we found an association of MDD with an increase of acid sphingomyelinase (ASM) activity. Thus, we asked whether chronic psychosocial stress as a detrimental factor contributing to the emergence of MDD would also affect ASM activity and sphingolipid (SL) metabolism. To induce chronic psychosocial stress in male mice we employed the chronic subordinate colony housing (CSC) paradigm and compared them to non-stressed single housed control (SHC) mice. We determined Asm activity in liver and serum, hepatic SL concentrations as well as hepatic mRNA expression of genes involved in SL metabolism. We found that hepatic Asm activity was increased by 28% (P = 0.006) and secretory Asm activity by 47% (P = 0.002) in stressed mice. C16:0-Cer was increased by 40% (P = 0.008). Gene expression analysis further revealed an increased expression of tumor necrosis factor (TNF)-alpha (P = 0.009) and of several genes involved in SL metabolism (Cers5, P = 0.028; Cers6, P = 0.045; Gba, P = 0.049; Gba2, P = 0.030; Ormdl2, P = 0.034; Smpdl3B; P = 0.013). Our data thus provides first evidence that chronic psychosocial stress, at least in mice, induces alterations in SL metabolism, which in turn might be involved in mediating the adverse health effects of chronic psychosocial stress and peripheral changes occurring in mood disorders.
Chronic psychosocial stress adversely affects human morbidity and is a risk factor for inflammatory disorders, liver diseases, obesity, metabolic syndrome, and major depressive disorder (MDD). In recent studies, we found an association of MDD with an increase of acid sphingomyelinase (ASM) activity. Thus, we asked whether chronic psychosocial stress as a detrimental factor contributing to the emergence of MDD would also affect ASM activity and sphingolipid (SL) metabolism. To induce chronic psychosocial stress in male mice we employed the chronic subordinate colony housing (CSC) paradigm and compared them to non-stressed single housed control (SHC) mice. We determined Asm activity in liver and serum, hepatic SL concentrations as well as hepatic mRNA expression of genes involved in SL metabolism. We found that hepatic Asm activity was increased by 28% (P = 0.006) and secretory Asm activity by 47% (P = 0.002) in stressed mice. C16:0-Cer was increased by 40% (P = 0.008). Gene expression analysis further revealed an increased expression of tumor necrosis factor (TNF)-alpha (P = 0.009) and of several genes involved in SL metabolism (Cers5, P = 0.028; Cers6, P = 0.045; Gba, P = 0.049; Gba2, P = 0.030; Ormdl2, P = 0.034; Smpdl3B; P = 0.013). Our data thus provides first evidence that chronic psychosocial stress, at least in mice, induces alterations in SL metabolism, which in turn might be involved in mediating the adverse health effects of chronic psychosocial stress and peripheral changes occurring in mood disorders.
Aims/hypothesis Low-protein diets are well known to improve glucose tolerance and increase energy expenditure. Increases in circulating fibroblast growth factor 21 (FGF21) have been implicated as a potential underlying mechanism. Methods We aimed to test whether low-protein diets in the context of a high-carbohydrate or high-fat regimen would also protect against type 2 diabetes in New Zealand Obese (NZO) mice used as a model of polygenetic obesity and type 2 diabetes. Mice were placed on high-fat diets that provided protein at control (16 kJ%; CON) or low (4 kJ%; low-protein/high-carbohydrate [LP/HC] or low-protein/high-fat [LP/HF]) levels. Results Protein restriction prevented the onset of hyperglycaemia and beta cell loss despite increased food intake and fat mass. The effect was seen only under conditions of a lower carbohydrate/fat ratio (LP/HF). When the carbohydrate/fat ratio was high (LP/HC), mice developed type 2 diabetes despite the robustly elevated hepatic FGF21 secretion and increased energy expenditure. Conclusion/interpretation Prevention of type 2 diabetes through protein restriction, without lowering food intake and body fat mass, is compromised by high dietary carbohydrates. Increased FGF21 levels and elevated energy expenditure do not protect against hyperglycaemia and type 2 diabetes per se.
Dendritic hPG-amid-C18-mPEG core-multishell nanocarriers (CMS) represent a novel class of unimolecular micelles that hold great potential as drug transporters, e. g., to facilitate topical therapy in skin diseases. Atopic dermatitis is among the most common inflammatory skin disorders with complex barrier alterations which may affect the efficacy of topical treatment. Here, we tested the penetration behavior and identified target structures of unloaded CMS after topical administration in healthy mice and in mice with oxazolone-induced atopic dermatitis. We further examined whole body distribution and possible systemic side effects after simulating high dosage dermal penetration by subcutaneous injection. Following topical administration, CMS accumulated in the stratum corneum without penetration into deeper viable epidermal layers. The same was observed in atopic dermatitis mice, indicating that barrier alterations in atopic dermatitis had no influence on the penetration of CMS. Following subcutaneous injection, CMS were deposited in the regional lymph nodes as well as in liver, spleen, lung, and kidney. However, in vitro toxicity tests, clinical data, and morphometry-assisted histopathological analyses yielded no evidence of any toxic or otherwise adverse local or systemic effects of CMS, nor did they affect the severity or course of atopic dermatitis. Taken together, CMS accumulate in the stratum corneum in both healthy and inflammatory skin and appear to be highly biocompatible in the mouse even under conditions of atopic dermatitis and thus could potentially serve to create a depot for anti-inflammatory drugs in the skin.
pH-sensitive nanoparticles have a great potential for dermal and transfollicular drug delivery. In this study, pH-sensitive, dexamethasone-loaded Eudragit (R) L 100, Eudragit (R) L 100-55, Eudragit (R) S 100, HPMCP-50, HPMCP-55 and cellulose acetate phthalate nanoparticles were prepared by nanoprecipitation and characterized. The pH-dependent swelling, erosion, dissolution and drug release kinetics were investigated in vitro using dynamic light scattering and Franz diffusion cells, respectively. Their toxicity potential was assessed by the ROS and MTT assays. 100-700 nm nanoparticles with high drug loading and entrapment efficiency were obtained. The nanoparticles bear no toxicity potential. Cellulose phthalates nanoparticles were more sensitive to pH than acrylates nanoparticles. They dissolved in 10 mM pH 7.5 buffer and released > 80% of the drug within 7 h. The acrylate nanoparticles dissolved in 40 mM pH 7.5 buffer and released 65-70% of the drug within 7 h. The nanoparticles remained intact in 10 and 40 mM pH 6.0 buffers (HPMCP nanoparticles dissolved in 40 mM pH 6.0 buffer) and released slowly. The nanoparticles properties could be modulated by blending the different polymers. In conclusion, various pH-sensitive nanoparticles that could release differently on the skin surface and dissolve and release in the hair follicles were obtained.
Improved knowledge of retinoblastoma chemotherapy resistance is needed to raise treatment efficiency. The objective of this study was to test whether etoposide alters glucosyl-ceramide, ceramide, sphingosine, and sphingosine-1-phosphate (sphingosine-1-P) levels in parental retinoblastoma cells (WERI Rb1) or their etoposide-resistant subclones (WERI EtoR). WERI Rb1 and WERI EtoR were incubated with 400 ng/ml etoposide for 24 h. Levels of glucosyl-ceramides, ceramides, sphingosine, sphingosine-1-P were detected by Q-TOF mass spectrometry. Statistical analysis was done by ANOVA followed by Tukey post-hoc test (p < 0.05). The mRNA expression of sphingolipid pathways enzymes in WERI Rb1, WERI EtoR and four human retinoblastoma tissue samples was analyzed by quantitative real-time PCR. Pathways enzymes mRNA expression confirmed similarities of human sphingolipid metabolism in both cell lines and tissue samples, but different relative expression. Significant up-regulation of sphingosine was seen in both cell lines (p < 0.001). Only sphingosine-1-P up-regulation was significantly increased in WERI EtoR (p < 0.01), but not in WERI Rb1 (p > 0.2). Both cell lines upregulate pro-apoptotic sphingosine after etoposide incubation, but only WERI EtoR produces additional survival favorable sphingosine-1-P. These data may suggest a role of sphingosine-1-P in retinoblastoma chemotherapy resistance, although this seems not to be the only resistance mechanism.
Background: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM.
Methods: Global DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study.
Results: Mothers with GDM displayed a significantly increased global placental DNA methylation (3.22 ± 0.63 vs. 3.00 ± 0.46 %; p = 0.013; ±SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1–4 (2.9–5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation.
Conclusions: This is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors.
A balanced sphingolipid rheostat is indispensable for dendritic cell function and survival and thus initiation of an immune response. Sphingolipid levels are dynamically maintained by the action of sphingolipid enzymes of which sphingosine kinases, S1P phosphatases (SGPP-1/2) and S1P lyase (SGPL-1), are pivotal in the balance of S1P and sphingosine levels. In this study, we present that SGPP-1 and SGPL-1 are regulated in inflammatory dendritic cells and contribute to S1P fate. TLR-dependent activation caused SGPL-1 protein downregulation with subsequent decrease of enzymatic activity by two-thirds. In parallel, confocal fluorescence microscopy revealed that endogenous SGPP-1 was expressed in nuclei of naive dendritic cells and was translocated into the cytoplasmatic compartment upon inflammatory stimulation resulting in dephosphorylation of S1P. Mass spectrometric determination showed that a part of the resulting sphingosine was released from the cell, increasing extracellular levels. Another route of diminishing intracellular S1P was possibly taken by its export via ATP-binding cassette transporter C1 which was upregulated in array analysis, while the S1P transporter, spinster homolog 2, was not relevant in dendritic cells. These investigations newly describe the sequential expression and localization of the endogenous S1P regulators SGPP-1 and SGPL-1 and highlight their contribution to the sphingolipid rheostat in inflammation.
Dendritic hPG-amid-C18-mPEG core-multishell nanocarriers (CMS) represent a novel class of unimolecular micelles that hold great potential as drug transporters, e. g., to facilitate topical therapy in skin diseases. Atopic dermatitis is among the most common inflammatory skin disorders with complex barrier alterations which may affect the efficacy of topical treatment.
Here, we tested the penetration behavior and identified target structures of unloaded CMS after topical administration in healthy mice and in mice with oxazolone-induced atopic dermatitis. We further examined whole body distribution and possible systemic side effects after simulating high dosage dermal penetration by subcutaneous injection.
Following topical administration, CMS accumulated in the stratum corneum without penetration into deeper viable epidermal layers. The same was observed in atopic dermatitis mice, indicating that barrier alterations in atopic dermatitis had no influence on the penetration of CMS. Following subcutaneous injection, CMS were deposited in the regional lymph nodes as well as in liver, spleen, lung, and kidney. However, in vitro toxicity tests, clinical data, and morphometry-assisted histopathological analyses yielded no evidence of any toxic or otherwise adverse local or systemic effects of CMS, nor did they affect the severity or course of atopic dermatitis.
Taken together, CMS accumulate in the stratum corneum in both healthy and inflammatory skin and appear to be highly biocompatible in the mouse even under conditions of atopic dermatitis and thus could potentially serve to create a depot for anti-inflammatory drugs in the skin.
Mutations in the gene encoding for filaggrin (FLG) are major predisposing factors for atopic dermatitis (AD). Besides genetic predisposition, immunological dysregulations considerably contribute to its pathophysiology. For example, thymic stromal lymphopoietin (TSLP) is highly expressed in lesional atopic skin and significantly contributes to the pathogenesis of AD by activating dendritic cells that then initiate downstream effects on, for example, T cells. However, little is known about the direct interplay between TSLP, filaggrin-deficient skin and other immune cells such as T lymphocytes. In the present study, FLG knockdown skin equivalents, characterised by intrinsically high TSLP levels, were exposed to activated CD4(+) T cells. T cell exposure resulted in an inflammatory phenotype of the skin equivalents. Furthermore, a distinct shift from a Th1/Th17 to a Th2/Th22 profile was observed following exposure of T cells to filaggrin-deficient skin equivalents. Interestingly, TSLP directly stimulated T cell migration exclusively in filaggrin-deficient skin equivalents even in the absence of dendritic cells, indicating a hitherto unknown role of TSLP in the pathogenesis of AD.
Nanoparticles can improve topical drug delivery: size, surface properties and flexibility of polymer nanoparticles are defining its interaction with the skin. Only few studies have explored skin penetration for one series of structurally related polymer particles with systematic alteration of material composition. Here, a series of rigid poly[acrylonitrile-co-(N-vinyl pyrrolidone)] model nanoparticles stably loaded with Nile Red or Rhodamin B, respectively, was comprehensively studied for biocompatibility and functionality. Surface properties were altered by varying the molar content of hydrophilic NVP from 0 to 24.1% and particle size ranged from 35 to 244 nm. Whereas irritancy and genotoxicity were not revealed, lipophilic and hydrophilic nanoparticles taken up by keratinocytes affected cell viability. Skin absorption of the particles into viable skin ex vivo was studied using Nile Red as fluorescent probe. Whilst an intact stratum corneum efficiently prevented penetration, almost complete removal of the horny layer allowed nanoparticles of smaller size and hydrophilic particles to penetrate into viable epidermis and dermis. Hence, systematic variations of nanoparticle properties allows gaining insights into critical criteria for biocompatibility and functionality of novel nanocarriers for topical drug delivery and risks associated with environmental exposure.
Novel nanogels that possess the capacity to change their physico-chemical properties in response to external stimuli are promising drug-delivery candidates for the treatment of severe skin diseases. As thermoresponsive nanogels (tNGs) are capable of enhancing penetration through biological barriers such as the stratum corneum and are taken up by keratinocytes of human skin, potential adverse consequences of their exposure must be elucidated. In this study, tNGs were synthesized from dendritic polyglycerol (dPG) and two thermoresponsive polymers. tNG_dPG_tPG are the combination of dPG with poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)) and tNG_dPG_pNIPAM the one with poly(N-isopropylacrylamide) (pNIPAM). Both thermoresponsive nanogels are able to incorporate high amounts of dexamethasone and tacrolimus, drugs used in the treatment of severe skin diseases. Cellular uptake, intracellular localization and the toxicological properties of the tNGs were comprehensively characterized in primary normal human keratinocytes (NHK) and in spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT). Laser scanning confocal microscopy revealed fluorescently labeled tNGs entered into the cells and localized predominantly within lysosomal compartments. MTT assay, comet assay and carboxy-H2DCFDA assay, demonstrated neither cytotoxic or genotoxic effects, nor any induction of reactive oxygen species of the tNGs in keratinocytes. In addition, both tNGs were devoid of eye irritation potential as shown by bovine corneal opacity and permeability (BCOP) test and red blood cell (RBC) hemolysis assay. Therefore, our study provides evidence that tNGs are locally well tolerated and underlines their potential for cutaneous drug delivery.
Within the last few decades, liquid chromatography-mass spectrometry (LC-MS) has become a preferred method for manifold issues in analytical biosciences, given its high selectivity and sensitivity. However, the analysis of fatty aldehydes, which are important components of cell metabolism, remains challenging. Usually, chemical derivatization prior to MS detection is required to enhance ionization efficiency. In this regard, the coupling of fatty aldehydes to hydrazines like 2,4-dinitrophenylhydrazine (DNPH) is a common approach. Additionally, hydrazones readily react with fatty aldehydes to form stable derivatives, which can be easily separated using high-performance liquid chromatography (HPLC) and subsequently detected by MS. Here, we exemplarily present the quantification of the long-chain fatty aldehyde (2E)-hexadecenal, a break-down product of the bioactive lipid sphingosine 1-phosphate (S1P), after derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) via isotope-dilution HPLC-electrospray ionization-quadrupole/time-of-flight (ESI-QTOF) MS. Moreover, we show that the addition of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC hydrochloride) as a coupling agent allows for simultaneous determination of fatty aldehydes and fatty acids as DAIH derivatives. Taking advantage of this, we describe in detail how to monitor the degradation of (2E)-hexadecenal and the concurrent formation of its oxidation product (2E)-hexadecenoic acid in lysates of human hepatoblastoma (HepG2) cells within this chapter.
Background/Aims:
Obesity is a main risk factor for the development of hepatic insulin resistance and it is accompanied by adipocyte hypertrophy and an elevated expression of different adipokines such as autotaxin (ATX). ATX converts lysophosphatidylcholine to lysophosphatidic acid (LPA) and acts as the main producer of extracellular LPA. This bioactive lipid regulates a broad range of physiological and pathological responses by activation of LPA receptors (LPA1-6).
Methods:
The activation of phosphatidylinositide 3-kinases (PI3K) signaling (Akt and GSK-3ß) was analyzed via western blotting in primary rat hepatocytes. Incorporation of glucose into glycogen was measured by using radio labeled glucose. Real-time PCR analysis and pharmacological modulation of LPA receptors were performed. Human plasma LPA levels of obese (BMI > 30, n = 18) and normal weight individuals (BMI 18.5-25, n = 14) were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS).
Results:
Pretreatment of primary hepatocytes with LPA resulted in an inhibition of insulin-mediated Gck expression, PI3K activation and glycogen synthesis. Pharmacological approaches revealed that the LPA3-receptor subtype is responsible for the inhibitory effect of LPA on insulin signaling. Moreover, human plasma LPA concentrations (16: 0 LPA) of obese participants (BMI > 30) are significantly elevated in comparison to normal weight individuals (BMI 18.5-25).
Conclusion:
LPA is able to interrupt insulin signaling in primary rat hepatocytes via the LPA3 receptor subtype. Moreover, the bioactive lipid LPA (16: 0) is increased in obesity.
Novel nanogels that possess the capacity to change their physico-chemical properties in response to external stimuli are promising drug-delivery candidates for the treatment of severe skin diseases. As thermoresponsive nanogels (tNGs) are capable of enhancing penetration through biological barriers such as the stratum corneum and are taken up by keratinocytes of human skin, potential adverse consequences of their exposure must be elucidated. In this study, tNGs were synthesized from dendritic polyglycerol (dPG) and two thermoresponsive polymers. tNG_dPG_tPG are the combination of dPG with poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)) and tNG_dPG_pNIPAM the one with poly(N-isopropylacrylamide) (pNIPAM). Both thermoresponsive nanogels are able to incorporate high amounts of dexamethasone and tacrolimus, drugs used in the treatment of severe skin diseases. Cellular uptake, intracellular localization and the toxicological properties of the tNGs were comprehensively characterized in primary normal human keratinocytes (NHK) and in spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (HaCaT). Laser scanning confocal microscopy revealed fluorescently labeled tNGs entered into the cells and localized predominantly within lysosomal compartments. MTT assay, comet assay and carboxy-H2DCFDA assay, demonstrated neither cytotoxic or genotoxic effects, nor any induction of reactive oxygen species of the tNGs in keratinocytes. In addition, both tNGs were devoid of eye irritation potential as shown by bovine corneal opacity and permeability (BCOP) test and red blood cell (RBC) hemolysis assay. Therefore, our study provides evidence that tNGs are locally well tolerated and underlines their potential for cutaneous drug delivery.
Preclinical assessment of penetration not only in intact, but also in barrier‐disrupted skin is important to explore the surplus value of novel drug delivery systems, which can be specifically designed for diseased skin. Here, we characterized physical and chemical barrier disruption protocols for short‐term ex vivo skin cultures with regard to structural integrity, physiological and biological parameters. Further, we compared the penetration of dexamethasone (Dex) in different nanoparticle‐based formulations in stratum corneum, epidermis and dermis extracts of intact vs. barrier‐disrupted skin as well as by dermal microdialysis at 6, 12 and 24 hours after topical application. Dex was quantified by liquid‐chromatography ‐ tandem‐mass spectrometry (LC‐MS/MS). Simultaneously, we investigated the Dex efficacy by interleukin (IL) analysis. Tape‐stripping (TS) and 4 hours sodium lauryl sulfate 5 % (SLS) exposure were identified as highly effective barrier disruption methods assessed by reproducible transepidermal water loss (TEWL) changes and IL‐6/8 increase which was more pronounced in SLS‐treated skin. The barrier state has also a significant impact on the Dex penetration kinetics: for all formulations, TS highly increased dermal Dex concentration despite the fact that nanocrystals quickly and effectively penetrated both, intact and barrier‐disrupted skin reaching significantly higher dermal Dex concentration after 6 hours compared to Dex cream. The surplus value of encapsulation in ethyl cellulose nanocarriers could mostly be observed when applied on intact skin, in general showing a delayed Dex penetration. Estimation of cytokines was limited due to the trauma caused by probe insertion. In summary, ex vivo human skin is a highly interesting short‐term preclinical model for the analysis of penetration and efficacy of novel drug delivery systems.
The lipid hydrolase enzyme acid sphingomyelinase (ASM) is required for the conversion of the lipid cell membrane component sphingomyelin into ceramide. In cancer cells, ASM-mediated ceramide production is important for apoptosis, cell proliferation, and immune modulation, highlighting ASM as a potential multimodal therapeutic target. In this study, we demonstrate elevated ASM activity in the lung tumor environment and blood serum of patients with non-small cell lung cancer (NSCLC). RNAi-mediated attenuation of SMPD1 in human NSCLC cells rendered them resistant to serum starvation-induced apoptosis. In a murine model of lung adenocarcinoma, ASM deficiency reduced tumor development in a manner associated with significant enhancement of Th1-mediated and cytotoxic T-cell-mediated antitumor immunity. Our findings indicate that targeting ASM in NSCLC can act by tumor cell-intrinsic and-extrinsic mechanisms to suppress tumor cell growth, most notably by enabling an effective antitumor immune response by the host. (C) 2017 AACR.
Controlled delivery of corticosteroids using nanoparticles to the skin and corneal epithelium may reduce their side effects and maximize treatment effectiveness. Dexamethasone-loaded ethyl cellulose, Eudragit® RS and ethyl cellulose/Eudragit® RS nanoparticles were prepared by the solvent evaporation method. Dexamethasone release from the polymeric nanoparticles was investigated in vitro using Franz diffusion cells. Drug penetration was also assessed ex vivo using excised human skin. Nanoparticle toxicity was determined by MTT and H2DCFDA assays. Eudragit® RS nanoparticles were smaller and positively charged but had a lower dexamethasone loading capacity (0.3–0.7%) than ethyl cellulose nanoparticles (1.4–2.2%). By blending the two polymers (1:1), small (105 nm), positively charged (+37 mV) nanoparticles with sufficient dexamethasone loading (1.3%) were obtained. Dexamethasone release and penetration significantly decreased with decreasing drug to polymer ratio and increased when Eudragit® RS was blended with ethyl cellulose. Ex vivo, drug release and penetration from the nanoparticles was slower than a conventional cream. The nanoparticles bear no toxicity potentials except ethyl cellulose nanoparticles had ROS generation potential at high concentration. In conclusion, the nanoparticles showed great potential to control the release and penetration of corticosteroids on the skin and mucus membrane and maximize treatment effectiveness.
Acid sphingomyelinase mediates murine acute lung injury following transfusion of aged platelets
(2017)
Pulmonary complications from stored blood products are the leading cause of mortality related to transfusion. Transfusion-related acute lung injury is mediated by antibodies or bioactive mediators, yet underlying mechanisms are incompletely understood. Sphingolipids such as ceramide regulate lung injury, and their composition changes as a function of time in stored blood. Here, we tested the hypothesis that aged platelets may induce lung injury via a sphingolipid-mediated mechanism. To assess this hypothesis, a two-hit mouse model was devised. Recipient mice were treated with 2 mg/kg intraperitoneal lipopolysaccharide (priming) 2 h before transfusion of 10 ml/kg stored (1-5 days) platelets treated with or without addition of acid sphingomyelinase inhibitor ARC39 or platelets from acid sphingomyelinase-deficient mice, which both reduce ceramide formation. Transfused mice were examined for signs of pulmonary neutrophil accumulation, endothelial barrier dysfunction, and histological evidence of lung injury. Sphingolipid profiles in stored platelets were analyzed by mass spectrophotometry. Transfusion of aged platelets into primed mice induced characteristic features of lung injury, which increased in severity as a function of storage time. Ceramide accumulated in platelets during storage, but this was attenuated by ARC39 or in acid sphingomyelinase-deficient platelets. Compared with wild-type platelets, transfusion of ARC39-treated or acid sphingomyelinase-deficient aged platelets alleviated lung injury. Aged platelets elicit lung injury in primed recipient mice, which can be alleviated by pharmacological inhibition or genetic deletion of acid sphingomyelinase. Interventions targeting sphingolipid formation represent a promising strategy to increase the safety and longevity of stored blood products.
Objective: We aimed to identify the role of the enzyme acid sphingomyelinase in the aging of stored units of packed red blood cells (pRBCs) and subsequent lung inflammation after transfusion.
Summary Background Data: Large volume pRBC transfusions are associated with multiple adverse clinical sequelae, including lung inflammation. Microparticles are formed in stored pRBCs over time and have been shown to contribute to lung inflammation after transfusion.
Methods: Human and murine pRBCs were stored with or without amitriptyline, a functional inhibitor of acid sphingomyelinase, or obtained from acid sphingomyelinase-deficient mice, and lung inflammation was studied in mice receiving transfusions of pRBCs and microparticles isolated from these units.
Results: Acid sphingomyelinase activity in pRBCs was associated with the formation of ceramide and the release of microparticles. Treatment of pRBCs with amitriptyline inhibited acid sphingomyelinase activity, ceramide accumulation, and microparticle production during pRBC storage. Transfusion of aged pRBCs or microparticles isolated from aged blood into mice caused lung inflammation. This was attenuated after transfusion of pRBCs treated with amitriptyline or from acid sphingomyelinase-deficient mice.
Conclusions: Acid sphingomyelinase inhibition in stored pRBCs offers a novel mechanism for improving the quality of stored blood.
Background: Abdominal aortic aneurysm (AAA) is a deadly irreversible weakening and distension of the abdominal aortic wall. The pathogenesis of AAA remains poorly understood. Investigation into the physical and molecular characteristics of perivascular adipose tissue (PVAT) adjacent to AAA has not been done before and is the purpose of this study.
Methods and Results: Human aortae, periaortic PVAT, and fat surrounding peripheral arteries were collected from patients undergoing elective surgical repair of AAA. Control aortas were obtained from recently deceased healthy organ donors with no known arterial disease. Aorta and PVAT was found in AAA to larger extent compared with control aortas. Immunohistochemistry revealed neutrophils, macrophages, mast cells, and T-cells surrounding necrotic adipocytes. Gene expression analysis showed that neutrophils, mast cells, and T-cells were found to be increased in PVAT compared with AAA as well as cathepsin K and S. The concentration of ceramides in PVAT was determined using mass spectrometry and correlated with content of T-cells in the PVAT.
Conclusions: Our results suggest a role for abnormal necrotic, inflamed, proteolytic adipose tissue to the adjacent aneurysmal aortic wall in ongoing vascular damage.
The role of serum amyloid A and sphingosine-1-phosphate on high-density lipoprotein functionality
(2017)
The high-density lipoprotein (HDL) is one of the most important endogenous cardiovascular protective markers. HDL is an attractive target in the search for new pharmaceutical therapies and in the prevention of cardiovascular events. Some of HDL’s anti-atherogenic properties are related to the signaling molecule sphingosine-1-phosphate (S1P), which plays an important role in vascular homeostasis. However, for different patient populations it seems more complicated. Significant changes in HDL’s protective potency are reduced under pathologic conditions and HDL might even serve as a proatherogenic particle. Under uremic conditions especially there is a change in the compounds associated with HDL. S1P is reduced and acute phase proteins such as serum amyloid A (SAA) are found to be elevated in HDL. The conversion of HDL in inflammation changes the functional properties of HDL. High amounts of SAA are associated with the occurrence of cardiovascular diseases such as atherosclerosis. SAA has potent pro-atherogenic properties, which may have impact on HDL’s biological functions, including cholesterol efflux capacity, antioxidative and anti-inflammatory activities. This review focuses on two molecules that affect the functionality of HDL. The balance between functional and dysfunctional HDL is disturbed after the loss of the protective sphingolipid molecule S1P and the accumulation of the acute-phase protein SAA. This review also summarizes the biological activities of lipid-free and lipid-bound SAA and its impact on HDL function.
Selenium increases hepatic DNA methylation and modulates one-carbon metabolism in the liver of mice
(2017)
The average intake of the essential trace element selenium (Se) is below the recommendation in most European countries, possibly causing sub-optimal expression of selenoproteins. It is still unclear how a suboptimal Se status may affect health. To mimic this situation, mice were fed one of three physiologically relevant amounts of Se. We focused on the liver, the organ most sensitive to changes in the Se supply indicated by hepatic glutathione peroxidase activity. In addition, liver is the main organ for synthesis of methyl groups and glutathione via one-carbon metabolism. Accordingly, the impact of Se on global DNA methylation, methylation capacity, and gene expression was assessed. We observed higher global DNA methylation indicated by LINE1 methylation, and an increase of the methylation potential as indicated by higher S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio and by elevated mRNA expression of serine hydroxymethyltransferase in both or either of the Se groups. Furthermore, increasing the Se supply resulted in higher plasma concentrations of triglycerides. Hepatic expression of glycolytic and lipogenic genes revealed consistent Se dependent up-regulation of glucokinase. The sterol regulatory element-binding transcription factor 1 (Srebf1) was also up-regulated by Se. Both effects were confirmed in primary hepatocytes. In contrast to the overall Se-dependent increase of methylation capacity, the up-regulation of Srebf1 expression was paralleled by reduced local methylation of a specific CpG site within the Srebf1 gene. Thus, we provided evidence that Se-dependent effects on lipogenesis involve epigenetic mechanisms. (C) 2017 The Authors. Published by Elsevier Inc.
Engineered nanogels are of high value for a targeted and controlled transport of compounds due to the ability to change their chemical properties by external stimuli. As it has been indicated that nanogels possess a high ability to penetrate the stratum corneum, it cannot be excluded that nanogels interact with dermal dendritic cells, especially in diseased skin. In this study the potential crosstalk of the thermore-sponsive nanogels (tNGs) with the dendritic cells of the skin was investigated with the aim to determine the immunotoxicological properties of the nanogels. The investigated tNGs were made of dendritic polyglycerol (dPG) and poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)), as polymer conferring thermoresponsive properties. Although the tNGs were taken up, they displayed neither cytotoxic and genotoxic effects nor any induction of reactive oxygen species in the tested cells. Interestingly, specific uptake mechanisms of the tNGs by the dendritic cells were depending on the nanogels cloud point temperature (Tcp), which determines the phase transition of the nanoparticle. The study points to caveolae-mediated endocytosis as being the major tNGs uptake mechanism at 37 degrees C, which is above the Tcp of the tNGs. Remarkably, an additional uptake mechanism, beside caveolae-mediated endocytosis, was observed at 29 degrees C, which is the Tcp of the tNGs. At this temperature, which is characterized by two different states of the tNGs, macropinocytosis was involved as well. In summary, our study highlights the impact of thermoresponsivity on the cellular uptake mechanisms which has to be taken into account if the tNGs are used as a drug delivery system.
Background: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM. Methods: Global DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study. Results: Mothers with GDM displayed a significantly increased global placental DNA methylation (3.22 +/- 0.63 vs. 3.00 +/- 0.46 %; p = 0.013; +/- SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1-4 (2.9-5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation. Conclusions: This is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors.
Background/Aims: Preterm birth (PTB) and low birth weight (LBW) significantly influence mortality and morbidity of the offspring in early life and also have long-term consequences in later life. A better understanding of the molecular mechanisms of preterm birth could provide new insights regarding putative preventive strategies. Metabolomics provides a powerful analytic tool to readout complex interactions between genetics, environment and health and may serve to identify relevant biomarkers. In this study, the association between 163 targeted maternal blood metabolites and gestational age was investigated in order to find candidate biomarkers for PTB. Methods: Five hundred twenty-three women were included into this observational study. Maternal blood was obtained before delivery. The concentration of 163 maternal serum metabolites was measured by flow injection tandem mass spectrometry. To find putative biomarkers for preterm birth, a three-step analysis was designed: bivariate correlation analysis followed by multivariable regression analysis and a comparison of mean values among gestational age groups. Results: Bivariate correlation analysis showed that 2 acylcarnitines (C16:2, C2), 1 amino acids (xLeu), 8 diacyl-PCs (PCaaC36:4, PCaaC38:4, PCaaC38:5, PCaaC38:6, PCaaC40:4, PCaaC40:5, PCaaC40:6, PCaaC42:4), and 1 Acylalkyl-PCs (PCaeC40:5) were inversely correlated with gestational age. Multivariable regression analysis confounded for PTB history, maternal body mass index (BMI) before pregnancy, systolic blood pressure at the third trimester, and maternal body weight at the third trimester, showed that the diacyl-PC PCaaC38:6 was the only metabolite inversely correlated with gestational age. Conclusions: Maternal blood concentrations of PCaaC38:6 are independently associated with gestational age. (C) 2016 The Author(s) Published by S. Karger AG, Basel
Drug loaded dendritic core-multishell (CMS) nanocarriers are of especial interest for the treatment of skin diseases, owing to their striking dermal delivery efficiencies following topical applications. CMS nanocarriers are composed of a polyglycerol core, connected by amide-bonds to an inner alkyl shell and an outer methoxy poly(ethylene glycol) shell. Since topically applied nanocarriers are subjected to biodegradation, the application of conventional amide-based CMS nanocarriers (10-A-18-350) has been limited by the potential production of toxic polyglycerol amines. To circumvent this issue, three tailored ester-based CMS nanocarriers (10-E-12-350, 10-E-15-350, 10-E-18-350) of varying inner alkyl chain length were synthesized and comprehensively characterized in terms of particle size, drug loading, biodegradation and dermal drug delivery efficiency. Dexamethasone (DXM), a potent drug widely used for the treatment of inflammatory skin diseases, was chosen as a therapeutically relevant test compound for the present study. Ester-and amide-based CMS nanocarriers delivered DXM more efficiently into human skin than a commercially available DXM cream. Subsequent in vitro and in vivo toxicity studies identified CMS (10-E-15-350) as the most biocompatible carrier system. The anti-inflammatory potency of DXM-loaded CMS (10-E-15-350) nanocarriers was assessed in TNF alpha supplemented skin models, where a significant reduction of the pro-inflammatory cytokine IL-8 was seen, with markedly greater efficacy than commercial DXM cream. In summary, we report the rational design and characterization of tailored, biodegradable, ester-based CMS nanocarriers, and their subsequent stepwise screening for biocompatibility, dermal delivery efficiency and therapeutic efficacy in a top-down approach yielding the best carrier system for topical applications. (C) 2016 Elsevier B.V. All rights reserved.
pH-sensitive nanoparticles which release in a controlled fashion on the skin or dissolve in the hair follicle could significantly improve treatment effectiveness and make transfollicular drug delivery a success. Dexamethasone-loaded Eudragit L 100 nanoparticles were prepared by nanoprecipitation from an organic drug-polymer solution. Their toxicity potential was assessed using isolated human fibroblasts. pH-dependent swelling and erosion kinetics of the nanoparticles were investigated by dynamic light scattering and viscosity measurements and its effect on drug release was assessed in vitro with Franz diffusion cells. Stable, 100-550 nm-sized dexamethasone-loaded Eudragit L 100 nanoparticles with drug loading capacity and entrapment efficiency as high as 83% and 85%, respectively, were obtained by using polyvinyl alcohol as a stabilizer and ethanol as organic solvent The nanoparticles showed little or no toxicity on isolated normal human fibroblasts. Dexamethasone existed in the nanoparticles as solid solution or in amorphous form. The nanoparticles underwent extensive swelling and slow drug release in media with a low buffer capacity (as low as 10 mM) and a higher pH or at a pH close to the dissolution pH of the polymer (pH 6) and a higher buffer capacity. In 40 mM buffer and above pH 6.8, the nanoparticles eroded fast or dissolved completely and thus released the drug rapidly. pH-sensitive nanoparticles which potentially release in a controlled manner on the stratum corneum but dissolve in the hair follicle could be prepared. (C) 2016 Elsevier B.V. All rights reserved.
Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C-6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.
Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model, and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28% of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.
Understanding penetration not only in intact, but also in lesional skin with impaired skin barrier function is important, in order to explore the surplus value of nanoparticle-based drug delivery for anti-inflammatory dermatotherapy. Herein, short-termex vivo cultures of (i) intact human skin, (ii) skin pretreated with tape-strippings and (iii) skin pre-exposed to sodium lauryl sulfate (SLS) were used to assess the penetration of dexamethasone (Dex). Intradermal microdialysis was utilized for up to 24 h after drug application as commercial cream, nanocrystals or ethyl cellulose nanocarriers applied at the therapeutic concentration of 0.05%, respectively. In addition, Dex was assessed in culture media and extracts from stratum corneum, epidermis and dermis after 24 h, and the results were compared to those in heat-separated split skin from studies in Franz diffusion cells. Providing fast drug release, nanocrystals significantly accelerated the penetration of Dex. In contrast to the application of cream and ethyl cellulose nanocarriers, Dex was already detectable in eluates after 6 h when applying nanocrystals on intact skin. Disruption of the skin barrier further accelerated and enhanced the penetration. Encapsulation in ethyl cellulose nanocarriers delayed Dex penetration. Interestingly, for all formulations highly increased concentrations in the dialysate were observed in tape-stripped skin, whereas the extent of enhancement was less in SLS-exposed skin. The results were confirmed in tissue extracts and were in line with the predictions made by in vitro release studies and ex vivo Franz diffusion cell experiments. The use of 45 kDa probes further enabled the collection of inflammatory cytokines. However, the estimation of glucocorticoid efficacy by Interleukin (IL)-6 and IL-8 analysis was limited due to the trauma induced by the probe insertion. Ex vivo intradermal microdialysis combined with culture media analysis provides an effective, skin-sparing method for preclinical assessment of novel drug delivery systems at therapeutic doses in models of diseased skin. (C) 2016 Elsevier B.V. All rights reserved.
Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect.
Reward-dependent instrumental behavior must continuously be re-adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re-learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction-induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re-learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid-focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM-ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane-located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction- and learning-related psychopathological conditions.
CD4(+) Foxp3(+) regulatory T cells (Tregs) depend on CD28 signaling for their survival and function, a receptor that has been previously shown to activate the acid sphingomyelinase (Asm)/ceramide system. In this article, we show that the basal and CD28-induced Asm activity is higher in Tregs than in conventional CD4(+) T cells (Tconvs) of wild-type (wt) mice. In Asm-deficient (Smpd1(-/-); Asm(-/-)) mice, as compared with wt mice, the frequency of Tregs among CD4(+) T cells, turnover of the effector molecule CTLA-4, and their suppressive activity in vitro were increased. The biological significance of these findings was confirmed in our Treg-sensitive mouse model of measles virus (MV) CNS infection, in which we observed more infected neurons and less MV-specific CD8(+) T cells in brains of Asm(-/-) mice compared with wt mice. In addition to genetic deficiency, treatment of wt mice with the Asm inhibitor amitriptyline recapitulated the phenotype of Asm-deficient mice because it also increased the frequency of Tregs among CD4(+) T cells. Reduced absolute cell numbers of Tconvs after inhibitor treatment in vivo and extensive in vitro experiments revealed that Tregs are more resistant toward Asm inhibitor-induced cell death than Tconvs. Mechanistically, IL-2 was capable of providing crucial survival signals to the Tregs upon inhibitor treatment in vitro, shifting the Treg/Tconv ratio to the Treg side. Thus, our data indicate that Asm-inhibiting drugs should be further evaluated for the therapy of inflammatory and autoimmune disorders.
Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect.