In human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by β-oxidation. Aim of the present study was to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI for different times (8d or 21d of culture) were used as comparison. OCTN2 transporter was equally expressed in both models and functional at the basolateral side. ATB0,+ was, instead, highly expressed and active on the apical membrane of EpiAirway™ and only in early-cultures of Calu-3 (8d but not 21d ALI). In both cell models, L-carnitine uptake on the apical side was significantly inhibited by the bronchodilators glycopyrrolate and tiotropium, that hence can be considered substrates of ATB0,+; ipratropium was instead effective on the basolateral side, indicating its interaction with OCTN2. Inflammatory stimuli, such as LPS or TNFα, caused an induction of SLC6A14/ATB0,+ expression in Calu-3 cells, along with a 2-fold increase of L-carnitine uptake only at the apical side; on the contrary SLC22A5/OCTN2 was not affected. As both OCTN2 and ATB0,+, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the study of drug inhalation and pulmonary delivery.

Alveolar epithelium, besides exerting a key role in gas exchange and surfactant production, plays important functions in host defense and inflammation. Pathological conditions associated to alveolar dysfunction include Acute Respiratory Distress Syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The use of predictive in vitro models of human alveolar epithelium is nowadays required for the study of disease mechanisms, as well as of pharmacokinetic parameters of pulmonary drugs delivery. Here, we employed a novel 3D model of human alveoli, namely EpiAlveolar™, consisting of primary alveolar epithelial cells, pulmonary endothelial cells and fibroblasts, that reflects properly the in vivo-like conditions. In EpiAlveolar™ we performed a characterization of Organic Cation Transporters (OCTs and OCTNs) expression and activity and we found that OCTN2, OCT1 and OCT3 are expressed on the basolateral membrane; instead, ATB0,+ transporter for cationic and neutral amino acids, which shares with OCTN2 the affinity for carnitine as substrate, is readily detectable and functional at the apical side. We also show that these transporters differentially interact with anticholinergic drugs. Overall, our findings reveal close similarities of EpiAlveolar™ with the tracheal/bronchial epithelium (EpiAirway™ model) and entrust this alveolar tissue as a potential tool for the screening of biopharmaceuticals molecules.

Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may delay or prevent the onset of Alzheimer's disease (AD). A subset of NSAIDs, including flurbiprofen, has been shown to selectively inhibit the production of beta-amyloid(1-42) (Abeta42), independently from their cyclooxygenase (COX) inhibiting activity. We evaluated the in vitro and in vivo profiles of CHF5022 and CHF5074, two flurbiprofen analogues. The in vitro Abeta inhibiting activity was evaluated in a human neuroglioma cell line (H4) carrying the double Swedish mutation (K595N/M596L) of the human amyloid precursor protein (APPsw). The in vitro anti-COX activity was evaluated using human recombinant enzymes isolated from transfected Sf-9 cells. The in vivo pharmacokinetic and pharmacodynamic profiles of the two compounds were evaluated in young APPsw transgenic mice (Tg2576) after oral gavage (100 or 300mgkg(-1) day(-1) for 4-5 days) and after medicated diet (375ppm for 4 weeks). R-Flurbiprofen was used as comparator. In vitro, CHF5022 and CHF5074 were found to be 3- and 7-fold more potent than R-flurbiprofen in inhibiting Abeta42 secretion (IC(50)s of 92, 40 and 268microM, respectively). Differently from R-flurbiprofen, CHF5022 and CHF5074 did not affect COX-1 (at 100microM) and COX-2 (at 300microM) activity. Similarly to R-flurbiprofen, no significant alteration in the expression profile of a subset of Notch intracellular domain-responsive genes was observed with either CHF5022 or CHF5074. In Tg2576 mice, CHF5022 was well tolerated when administered by oral gavage (100mgkg(-1) day(-1) for 5 days) or by medicated diet (56mg kg(-1) day(-1) for 4 weeks). R-Flurbiprofen was poorly tolerated in the diet (32mgkg(-1) day(-1)) with 55% of the animals dying during the first week of treatment. After 4-5 days of oral gavage, CHF5022 and CHF5074 plasma and brain levels at 3h were found to increase with the dose, leading to brain concentrations of about 10% and 5% of the corresponding plasma concentrations, respectively. In animals fed for 4 weeks with compound-supplemented diet, mean plasma (580microM) and brain (20microM) Cyrillic) concentrations of CHF5022 were 8 and 15 times higher than those of R-flurbiprofen. Plasma Abeta42 concentration was dose-dependently decreased by CHF5022 and CHF5074. Brain Abeta levels (formic acid-extractable) were not significantly affected by either compound, although Abeta42 levels tended to inversely correlate (P=0.105) with CHF5022 concentration in the brain. CHF5022 and CHF5074 thus appear to have a promising in vitro and in vivo profile. This warrants further evaluation of their long-term effects on Abeta brain pathology.

Lysinuric protein intolerance (LPI) is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y+L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA), a significant induction of the expression and release of the inflammatory mediators IL1β and TNFα was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1β has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation.

The ATP-binding cassette (ABC) transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) play a crucial role in the translocation of a broad range of drugs; data about their expression and activity in lung tissue are controversial. Here, we address their expression, localization and function in EpiAirway™, a three-dimensional (3D)-model of human airways; Calu-3 cells, a representative in vitro model of bronchial epithelium, are used for comparison. Transporter expression has been evaluated with RT-qPCR and Western blot, the localization with immunocytochemistry, and the activity by measuring the apical-to-basolateral and basolateral-to-apical fluxes of specific substrates in the presence of inhibitors. EpiAirway™ and Calu-3 cells express high levels of MRP1 on the basolateral membrane, while they profoundly differ in terms of BCRP and MDR1: BCRP is detected in EpiAirway™, but not in Calu-3 cells, while MDR1 is expressed and functional only in fully-differentiated Calu-3; in EpiAirway™, MDR1 expression and activity are undetectable, consistently with the absence of the protein in specimens from human healthy bronchi. In summary, EpiAirway™ appears to be a promising tool to study the mechanisms of drug delivery in the bronchial epithelium and to clarify the role of ABC transporters in the modulation of the bioavailability of administered drugs.

ATP-binding cassette (ABC) transporters are a large superfamily of membrane transporters that facilitate the translocation of different substrates. While ABC transporters are clearly expressed in various tumor cells where they can play a role in drug extrusion, the presence of these transporters in normal lung tissues is still controversial. Here, we performed an analysis of ABC transporters in EpiAlveolarTM, a recently developed model of human alveoli, by defining the expression and activity of MDR1, BCRP, and MRPs. Immortalized primary epithelial cells hAELVi (human alveolar epithelial lentivirus-immortalized cells) were employed for comparison. Our data underline a close homology between these two models, where none of the ABC transporters here studied are expressed on the apical membrane and only MRP1 is clearly detectable and functional at the basolateral side. According to these findings, we can conclude that other thus-far-unidentified transporter/s involved in drug efflux from alveolar epithelium deserve investigations.

Organic cation transporters (OCTs) and novel organic cation transporters (OCTNs) are responsible for drug delivery in the intestine and kidney; in the lung, OCTs mediate inhaled drugs' transport, although their physiological role in airways remains poorly understood. The studies addressing OCTs/OCTNs in human airways were mostly performed in immortal or transformed cell lines; here, we studied OCTs in EpiAirway™, a recently developed in vitro model of normal bronchial epithelium. Calu-3 monolayers were used for comparison. The activity of OCTs was evaluated by measuring the uptake of 1-methyl-4-phenylpyridinium (MPP+) at the apical and basolateral side of monolayers and protein expression through Western Blot analysis. OCTs and OCTNs expression, along with that of Amino acid Transporter B0,+ (ATB0,+)transporter, was determined by measuring the number of mRNA molecules through quantitative Polymerase Chain Reaction (qPCR). The interaction of the transporters with bronchodilators was also assessed. Results highlight significant differences between Calu-3 cells and EpiAirway™, since, in the latter, OCTs are active only on the basolateral membrane where they interact with the bronchodilator ipratropium. No activity of OCTs is detectable at the apical side; there, the most abundant carrier is, instead, SLC6A14/ATB0,+, that can thus be potentially listed among organic cation transporters responsible for drug delivery in the lung.

Recently, mesenchymal stromal stem cells (MSCs) have been proposed as therapeutic agents because of their promising preclinical features and good safety profile. However, their introduction into clinical practice has been associated with a suboptimal therapeutic profile. In this review, we address the biodistribution of MSCs in preclinical studies with a focus on the current understanding of the pharmacodynamics (PD) and pharmacokinetics (PK) of MSCs as key aspects to overcome unsatisfactory clinical benefits of MSC application. Beginning with evidence of MSC biodistribution and highlighting PK and PD factors, a new PK-PD model is also proposed. According to this theory, MSCs and their released factors are key players in PK, and the efficacy biomarkers are considered relevant for PD in more predictive preclinical investigations. Accounting for the PK-PD relationship in MSC translational research and proposing new models combined with better biodistribution studies could allow realization of the promise of more robust MSC clinical translation.

Mass spectrometry (MS)-based proteomics has recently attracted the attention from forensic pathologists. This work is the first report of the development of a shotgun bottom-up proteomic approach based on rapid protein extraction and nano-liquid chromatography/high-resolution mass spectrometry applied to full-thickness human skin for the differential analysis of normal and ecchymotic tissues to identify new biomarkers for bruise characterization and dating. We identified around 2000 proteins from each pooled extract. The method showed excellent precision on independent replicates, with Pearson correlation coefficients always higher than 95%. Glycophorin A, a known biomarker of vital wounds from immunochemical studies, was identified only in ecchymotic tissues, as confirmed by Western blotting analysis. This finding suggests that this protein can be used as a MS-detectable biomarker of wound vitality. By focusing on skin samples from individuals with known wound dating, besides Glycophorin A, other proteins differentially expressed in ecchymotic samples and dependant on wound age were identified, although further analysis on larger datasets are needed to validate these findings. This study paves the way for an in-depth investigation of the potential of MS-based techniques for wound examination in forensic pathology, overcoming the limitations of immunochemical assays.