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Purpose: Exposure to hypoxia has been suggested to activate multiple adaptive pathways so that muscles are better able to maintain cellular energy homeostasis. However, there is limited research regarding the tissue specificity of this response. The aim of this study was to investigate the influence of tissue specificity on mitochondrial adaptations of rat skeletal and heart muscles after 4 weeks of normobaric hypoxia (FiO2: 0.10). Methods: Twenty male Wistar rats were randomly assigned to either normobaric hypoxia or normoxia. Mitochondrial respiration was determined in permeabilised muscle fibres from left and right ventricles, soleus and extensorum digitorum longus (EDL). Citrate synthase activity and the relative abundance of proteins associated with mitochondrial biogenesis were also analysed. Results: After hypoxia exposure, only the soleus and left ventricle (both predominantly oxidative) presented a greater maximal mass-specific respiration (+48 and +25%, p < 0.05) and mitochondrial-specific respiration (+75 and +28%, p < 0.05). Citrate synthase activity was higher in the EDL (0.63 ± 0.08 vs 0.41 ± 0.10 µmol min− 1 µg− 1) and lower in the soleus (0.65 ± 0.17 vs 0.87 ± 0.20 µmol min− 1 µg− 1) in hypoxia with respect to normoxia. There was a lower relative protein abundance of PGC-1α (−25%, p < 0.05) in the right ventricle and a higher relative protein abundance of PGC-1β (+43%, p < 0.05) in the left ventricle of rats exposed to hypoxia, with few differences for protein abundance in the other muscles. Conclusion: Our results show a muscle-specific response to 4 weeks of normobaric hypoxia. Depending on fibre type, and the presence of ventricular hypertrophy, muscles respond differently to the same degree of environmental hypoxia

Purpose Nanotechnologies turned out to be promising in the development of diagnostic and therapeutic approaches toward neurodegenerative disorders. However, only a very scant number of nanodevices until now proved to be effective on preclinical animal models. Although specific tests in vivo are available to assess the potential toxicity of these nanodevices on cognitive functions, those to evaluate their biosafety in vitro on neurons are still to be improved. Materials and methods We utilized the patch-clamp technique on primary cultures of cortical neural cells isolated from neonatal rats, aiming to evaluate their electrical properties after the incubation with liposomes (mApoE-PA-LIPs), previously proved able to cross the blood–brain barrier and to be effective on mouse models of Alzheimer’s disease (AD), both in the absence and in the presence of β-amyloid peptide oligomers. Results Data show a high degree of biocompatibility, evaluated by lactate dehydrogenase (LDH) release and MTT assay, and the lack of cellular internalization. After the incubation with mApoE-PA-LIPs, neuronal membranes show an increase in the input resistance (from 724.14±76 MΩ in untreated population to 886.06±86 MΩ in the treated one), a reduction in the rheobase current (from 29.6±3 to 24.2±3 pA in untreated and treated, respectively), and an increase of the firing frequency, consistent with an ultimate increase in intrinsic excitability. Data obtained after co-incubation of mApoE-PA-LIPs with β-amyloid peptide oligomers suggest a retention of liposome efficacy. Conclusion These data suggest the ability of liposomes to modulate neuronal electrical properties and are compatible with the previously demonstrated amelioration of cognitive functions induced by treatment of AD mice with liposomes. We conclude that this electrophysiological approach could represent a useful tool for nanomedicine to evaluate the effect of nanoparticles on intrinsic neuronal excitability.

Purpose: Chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLN) are a new class of nanodevices to effectively deliver anti-cancer drugs to tumoral cells. This study investigated their antitumoral effects ‘per se’, using a mathematical model validated on experimental data. Methods: OLN were prepared and characterized either in vitro or in vivo. TUBO cells, established from a lobular carcinoma of a BALB-neuT mouse, were investigated following 48 h of incubation in the absence/presence of different concentrations of OLN. OLN internalization, cell viability, necrosis, apoptosis, cell cycle and reactive oxygen species (ROS) production were checked as described in the Method section. In vivo tumor growth was evaluated after subcutaneous transplant in BALB/c mice of TUBO cells either without treatment or after 24 h incubation with 10% v/v OLN. Results: OLN showed sizes of about 350 nm and a positive surface charge (45 mV). Dose-dependent TUBO cell death through ROS-triggered apoptosis following OLN internalization was detected. A mathematical model predicting the effects of OLN uptake was validated on both in vitro and in vivo results. Conclusions: Due to their intrinsic toxicity OLN might be considered an adjuvant tool suitable to deliver their therapeutic cargo intracellularly and may be proposed as promising combined delivery system.

Isolated human airway smooth muscle (ASM) tissue contractility studies are essential for understanding the role of ASM in respiratory disease, but limited availability and cost render storage options necessary for optimal use. However, to our knowledge, no comprehensive study of cryopreservation protocols for isolated ASM has been performed to date. We tested several cryostorage protocols on equine trachealis ASM using different cryostorage media [1.8 M dimethyl sulfoxide and fetal bovine serum (FBS) or Krebs-Henseleit (KH)] and different degrees of dissection (with or without epithelium and connective tissues attached) before storage. We measured methacholine (MCh), histamine, and isoproterenol (Iso) dose-responses and electrical field stimulation (EFS) and MCh force-velocity curves. We confirmed our findings in human trachealis ASM stored undissected in FBS. Maximal stress response to MCh was decreased more in dissected than undissected equine tissues. EFS force was decreased in all equine but not in human cryostored tissues. Furthermore, in human cryostored tissues, EFS maximal shortening velocity was decreased, and Iso response was potentiated after cryostorage. Overnight incubation with 0.5 or 10% FBS did not recover contractility in the equine tissues but potentiated Iso response. Overnight incubation with 10% FBS in human tissues showed maximal stress recovery and maintenance of other contractile parameters. ASM tissues can be cryostored while maintaining most contractile function. We propose an optimal protocol for cryostorage of ASM as undissected tissues in FBS or KH solution followed by dissection of the ASM bundles and a 24-h incubation with 10% FBS before mechanics measurements.

Background Inhaled oxidative toxicants present in ambient air cause airway epithelial injury, inflammation, and airway hyperresponsiveness. Effective adaptation to such environmental insults is essential for the preservation of pulmonary function, whereas failure or incomplete adaptation to oxidative injury can render the host susceptible to the development of airway disease. Objective We sought to explore the mechanisms of airway adaptation to oxidative injury. Methods For a model to study pulmonary adaptation to oxidative stress–induced lung injury, we exposed mice to repeated nose-only chlorine gas exposures. Outcome measures were evaluated 24 hours after the last chlorine exposure. Lung mechanics and airway responsiveness to methacholine were assessed by using the flexiVent. Inflammation and antioxidant responses were assessed in both bronchoalveolar lavage fluid and lung tissue. Using both loss or gain of function and genomic approaches, we further dissected the cellular and molecular mechanisms involved in pulmonary adaptation. Results Repeated exposures to oxidative stress resulted in pulmonary adaptation evidenced by abrogation of neutrophilic inflammation and airway hyperresponsiveness. This adaptation was independent of antioxidant mechanisms and regulatory T cells but dependent on residential alveolar macrophages (AMs). Interestingly, 5% of AMs expressed forkhead box P3, and depletion of these cells abolished adaptation. Results from transcriptomic profiling and loss and gain of function suggest that adaptation might be dependent on TGF-β and prostaglandin E2. Conclusion Pulmonary adaptation during oxidative stress–induced lung injury is mediated by a novel subset of forkhead box P3–positive AMs that limits inflammation, favoring airway adaptation and host fitness through TGF-β and prostaglandin E2.

Antonina Orlando,1 Francesca Re,1 Silvia Sesana,1 Ilaria Rivolta,1 Alice Panariti,1 Davide Brambilla,2 Julien Nicolas,2 Patrick Couvreur,2 Karine Andrieux,2 Massimo Masserini,1 Emanuela Cazzaniga1 1Department of Health Sciences, University of Milano-Bicocca, Monza, Italy; 2Institut Galien Paris Sud, University Paris-Sud, Châtenay-Malabry, France Background: As part of a project designing nanoparticles for the treatment of Alzheimer’s disease, we have synthesized and characterized a small library of nanoparticles binding with high affinity to the β-amyloid peptide and showing features of biocompatibility in vitro, which are important properties for administration in vivo. In this study, we focused on biocompatibility issues, evaluating production of nitric oxide by cultured human umbilical vein endothelial cells and macrophages, used as models of cells which would be exposed to nanoparticles after systemic administration. Methods: The nanoparticles tested were liposomes and solid lipid nanoparticles carrying phosphatidic acid or cardiolipin, and PEGylated poly(alkyl cyanoacrylate) nanoparticles (PEG-PACA). We measured nitric oxide production using the Griess method as well as phosphorylation of endothelial nitric oxide synthase and intracellular free calcium, which are biochemically related to nitric oxide production. MTT viability tests and caspase-3 detection were also undertaken. Results: Exposure to liposomes did not affect the viability of endothelial cells at any concentration tested. Increased production of nitric oxide was detected only with liposomes carrying phosphatidic acid or cardiolipin at the highest concentration (120 µg/mL), together with increased synthase phosphorylation and intracellular calcium levels. Macrophages exposed to liposomes showed a slightly dose-dependent decrease in viability, with no increase in production of nitric oxide. Exposure to solid lipid nanoparticles carrying phosphatidic acid decreased viability in both cell lines, starting at the lowest dose (10 µg/mL), with increased production of nitric oxide detected only at the highest dose (1500 µg/mL). Exposure to PEG-PACA affected cell viability and production of nitric oxide in both cell lines, but only at the highest concentration (640 µg/mL). Conclusion: Liposomal and PEG-PACA nanoparticles have a limited effect on vascular homeostasis and inflammatory response, rendering them potentially suitable for treatment of Alzheimer’s disease. Moreover, they highlight the importance of testing such nanoparticles for production of nitric oxide in vitro in order to identify a therapeutic dose range suitable for use in vivo. Keywords: nanoparticles, nitric oxide, endothelial cells, macrophages, Alzheimer’s disease

Perfluoropentane (PFP)-based oxygen-loaded nanobubbles (OLNBs) were previously proposed as adjuvant therapeutic tools for pathologies of different etiology sharing hypoxia as a common feature, including cancer, infection, and autoimmunity. Here we introduce a new platform of oxygen nanocarriers, based on 2H,3H-decafluoropentane (DFP) as core fluorocarbon. These new nanocarriers have been named oxygen-loaded nanodroplets (OLNDs) since DFP is liquid at body temperature, unlike gaseous PFP. Dextran-shelled OLNDs, available either in liquid or gel formulations, display spherical morphology, ~600 nm diameters, anionic charge, good oxygen carrying capacity, and no toxic effects on human keratinocytes after cell internalization. In vitro OLNDs result more effective in releasing oxygen to hypoxic environments than former OLNBs, as demonstrated by analysis through oxymetry. In vivo, OLNDs effectively enhance oxy-hemoglobin levels, as emerged from investigation by photoacoustic imaging. Interestingly, ultrasound (US) treatment further improves transdermal oxygen release from OLNDs. Taken together, these data suggest that US-activated, DFP-based OLNDs might be innovative, suitable and cost-effective devices to topically treat hypoxia-associated pathologies of the cutaneous tissues.

Communication between cardiomyocytes depends upon gap junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylase (HAT) and deacetylase (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5. Hz for 24. h significantly reduced connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation.In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43.

Rationale Chlorine gas (Cl2) is a potent oxidant and trigger of irritant induced asthma. We explored NF-E2–related factor 2 (Nrf2)-dependent mechanisms in the asthmatic response to Cl2, using Nrf2-deficient mice, buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and sulforaphane (SFN), a phytochemical regulator of Nrf2. Methods Airway inflammation and airway hyperresponsiveness (AHR) were assessed 24 and 48 h after a 5-min nose-only exposure to 100 ppm Cl2 of Nrf2-deficient and wild type Balb/C mice treated with BSO or SFN. Animals were anesthetized, paralyzed and mechanically ventilated (FlexiVent™) and challenged with aerosolized methacholine. Bronchoalveolar lavage (BAL) was performed and lung tissues were harvested for assessment of gene expression. Results Cl2 exposure induced a robust AHR and an intense neutrophilic inflammation that, although similar in Nrf2-deficient mice and wild-type mice at 24 h after Cl2 exposure, were significantly greater at 48 h post exposure in Nrf2-deficient mice. Lung GSH and mRNA for Nrf2-dependent phase II enzymes (NQO-1 and GPX2) were significantly lower in Nrf2-deficient than wild-type mice after Cl2 exposure. BSO reduced GSH levels and promoted Cl2-induced airway inflammation in wild-type mice, but not in Nrf2-deficient mice, whereas SFN suppressed Cl2-induced airway inflammation in wild-type but not in Nrf2-deficient mice. AHR was not affected by either BSO or SFN at 48 h post Cl2 exposure. Conclusions Nrf2-dependent phase II enzymes play a role in the resolution of airway inflammation and AHR after Cl2 exposure. Moderate deficiency of GSH affects the magnitude of acute inflammation but not AHR.

This study analyzed the time dependence decay of the mRNA of selected genes important for the hypoxia response. The genes chosen were the two isoforms of hypoxia-inducible factors, the three isoforms of the prolyl hydroxylase domain protein, the vascular endothelial growth factor and endothelial nitric oxide synthase. mRNA and proteins were extracted from lungs obtained from control, hypoxic and 15 minutes normoxic recovered rats and analyzed by Real-time RT-PCR or by the Western Blot technique. Results indicated that in normoxia isoform 2á was the more represented hypoxia-inducible factor mRNA, and among the prolyl hydroxylase domain transcripts, isoform 3 was the least abundant. Moreover, in chronic hypoxia only hypoxia-inducible factor 1α and prolyl hydroxylase domain protein 3 increased significantly, while after 15 minutes of recovery all the mRNAs tested were decreased except endothelial nitric oxide synthase mRNA. In terms of proteins, hypoxia-inducible 1α was the isoform more significant in the nucleus, while 2á predominated in the cytosol. While the former was steady even after a brief recovery from hypoxia, the latter underwent a strong degradation. In conclusion we showed the relevance of the decay in the mRNA and protein levels upon re-oxygenation in normoxia. We believe that this has to be considered in research studies dealing with recovery from hypoxia.

The aim of present work was to elucidate the interaction of solid lipid nanoparticles (SLNs) with cellular plasma-membrane to gain insight of intracellular drug delivery. To this aim we followed the uptake of coumarin-6 (a drug model) either free in the extracellular medium or loaded on SLN (c-SLN). Alveolar epithelial cells were exposed to a biocompatible concentration of c-SLN (0.01 mg/ml of tripalmitin) prepared by warm microemulsion whose lipid matrix was constituted by low melting point molecules (fatty acids, triglycerides). Intracellular fluorescence and preferential accumulation in the perinuclear region were increased by 54.8% on comparing c-SLN to the same amount of free coumarin-6 in the medium. Lowering temperature from 37 ° to 4 °C decreased the intracellular signal intensity by about 48% equally for the free as well as for loaded drug, thus suggesting the inhibition of a similar non-endocytotic entrance pathway. No specific co-localization of the fluorescence with intracellular organelles was found. The c-SLN calorimetric profile obtained with differential scanning calorimetry (DSC), revealing transition within the range 58-62 °C, altered remarkably upon incubation with cells, suggesting a change in SLN structure after association with cells membranes. We propose that the uptake of the model drug loaded on SLN is only partly related to the endocytotic pathway; it occurs despite the loss of integrity of the original SLN structure and it appears to be more efficient when the drug is vehicled rather than being free in the culture medium.

Colchicine is a naturally occurring alkaloid used in human and veterinary medicine. It shows genotoxicity in in vitro and in vivo systems even at low concentrations. Therefore, no ADI has been established, and colchicine has been included in Annex IV of Council Regulation (EEC) No. 2377/90. No abuse of this drug in intensive livestock farming has yet been reported. However, there may be a natural route of entry for this compound into the food chain when Colchicum autumnale is consumed by animals kept outdoors. To address this concern, we developed and validated a highly sensitive and selective quantitative LC-ESI-MS-MS method for the detection of colchicine in sheep serum and milk. For sample pretreatment, all samples were liquid-liquid extracted with phosphate buffer (pH 8.0) and dichloromethane. LC separation was carried out on an RP C18 column employing a 0.5% formic acid/acetonitrile gradient system. The recoveries in both matrixes at a concentration range from 0.0005 to 1 mg/L were80% with RSDs of10%. The applicability of the method was demonstrated in serum and the corresponding milk samples from Albanian sheep. After exposure to C. autumnale, the animals showed clinical signs of intoxication. Significant concentrations (up to 1 mg/L) of the drug were found in serum as well as in milk samples.

This paper presents monitoring data on levels of aflatoxin M1 in the farm-gate milk in Albania. The monitoring included 120 evenly distributed samples collected in winter and summer from various farms all over the country. The levels of aflatoxin M1 were determined using the quantitative thin layer chromatography. On average, the winter milk samples revealed higher concentrations of aflatoxin M1 than the summer samples. Thirteen percent of the winter samples resulted above the 0.5 microgram/kg level, as compared to 3% of the summer samples exceeding that level. Skimmed and semi-skimmed milk from the same source contained comparatively lower levels of aflatoxin M1 than whole milk.

In April 1997, a severe lead poisoning in humans was reported in rural Albania and a 30-y-old male and a 17-y-old female died. Twenty-three persons exhibiting signs of lead intoxication recovered following intensive EDTA-Ca chelation therapy. The poisoning resulted from lead contaminated flour originating from repair of a mill's cracked grindstone with metallic lead. Lead levels in flour samples averaged 325 +/- 18 ppm and in bran were 370 +/- 22 ppm. Blood lead levels in intoxicated individuals averaged 0.42 +/- 0.05 ppm and were compatible with their symptomatic status. Contaminated bran from the same mill was fed to lactating cows as well. Lead levels in blood and milk of exposed cows averaged 0.27 +/- 0.06 and 0.61 +/- 0.04 ppm, respectively.

Pyrrolizidine alkaloids (PAs) contamination of animal products, may pose a threat to human health. Among ruminants, sheep appear to be more resistant to PAs poisoning and consequently comparatively higher concentrations of the PAs into their milk might be expected. To investigate the PAs transfer into sheep's milk following multiple intakes, three lactating sheep were submitted to the administration of 14C-seneciphylline in daily amounts of 32.4 mg, for a 5 days period. The appearance of radioactivity, derived from this compound was monitored in blood and milk and was calculated as seneciphylline, Alkaloid levels in blood during the first hour following the first dose administration, were 167 ng/ml and reached a maximum of 518 ng/ml at the 5th day of multiple treatment, before falling below the detection limit at the second day of the post treatment period. The Milk levels were respectively 62.3 ng/ml at the 3rd hour following the first dose administration, reaching a maximum of 987 ng/ml at the 5th day and falling below the detection limit at the fourth day of the postreatment period. The Seneciphylline levels in liver at the end of the 5 d multiple administration period averaged at 85 ng/g corresponding to 0.22% of the daily intake.

The present communication describes the use of a quantitative thin-layer-chromatography method for the determination of colchicine in milk of a lactating sheep following a 10 mg single oral intake of the alkaloid extracted from the leaves of Colchicum autumnale. The concentration of the alkaloid in the leaves was 0,18% by weight. Unchanged colchicine can be detected in milk with the described method within the first 6 hours following the intake, whereas the maximal concentration of the alkaloid is reached 9 hours after the administration. Almost 40 hours following the intake the detection of the alkaloid in milk is not more possible. Postmortal determinations of the tissue levels of the alkaloid revealed major concentrations in the bone marrow, whereas only trace amounts were identified in heart and muscular tissue.

"Peak" levels of radioiodine (131J) in sheep and goats milk were reached between 12-24 hrs following their contamination. At the end of the nine days period for sheep and of 12 days period for goats, following a single contamination with the prescribed 131J dose, the radioiodine level is 0,25% of the contamination dose. Following a multiple biweekly radioiodine contamination, the corresponding radioactivity levels in milk are much higher than in blood. From the fourth day on, the ratio between the respective levels in milk and blood remains constant. Following a biweekly multiple contamination in goats, average concentration of 131J in thyroid is about 220% of the daily contamination dose, whereas the corresponding concentrations in milk and blood are 33% and 5% of the daily contamination dose. The corresponding values in sheep are 140% for thyroid and 26% and 3.5% for milk and blood. Chemicals used as "iodine metabolic inhibitors" do sensibly reduce radioiodine levels in milk, while simultaneously increasing the corresponding levels in blood.