Mucus-producing epithelial models for investigating the activity of gene delivery systems in the lung.

Inhaled transfection particles have to penetrate the mucus layer lining the airways to successfully deliver their therapeutic nucleic acid payload to target cells in the underlying epithelium. However, the in vitro models used for evaluating gene carrier efficiency often disregard this viscous defensive barrier. In this study, the two mucus-secreting cell lines NCI-H292 and Calu-3 were selected to develop a series of epithelial models displaying gradual mucus production. In NCI-H292 models, a gradual increase in the MUC5AC mucin was obtained after cell exposure to inducers. In Calu-3 models, MUC5AC production increased as a function of culture duration (3, 7, 14 days) at the air-liquid interface (ALI). Six DOPC-derived cationic lipids were designed and their pDNA delivery activity was evaluated to validate these cellular models. The strongest impairment of the lipid delivery activity was observed in the Calu-3 14-d ALI model. The MUC5AC production in this model was the greatest and the mucus layer was 20 µm thick. The mucus exhibited a solid viscoelastic behavior, and represented a major hindrance to lipoplex diffusion. The Calu-3 14-d ALI model will be highly useful for accurate evaluation of gene carriers intended for airway administration and characterization of their interactions with the mucus.

IgE recognition of the house dust mite allergen Der p 37 is associated with asthma.

BACKGROUND: House dust mite (HDM) allergens are major elicitors of allergic reactions worldwide. OBJECTIVE: Identification, characterization, and evaluation of diagnostic utility of a new important HDM allergen was performed. METHODS: A cDNA coding for a new Dermatophagoides pteronyssinus (Dp) allergen, Der p 37, was isolated from a Dp expression library with allergic patients' IgE antibodies. Recombinant Der p 37 (rDer p 37) expressed in Escherichia coli was purified, then characterized by mass spectrometry, circular dichroism, and IgE reactivity by ImmunoCAP ISAC technology with sera from 111 clinically defined HDM-allergic patients. The allergenic activity of rDer p 37 was studied by basophil activation and CD4+ T-cell responses by carboxyfluorescein diacetate succinimidyl ester dilution assays. Specific antibodies raised against rDer p 37 were used for the ultrastructural localization of Der p 37 in mites by immunogold transmission electron microscopy. RESULTS: Der p 37, a 26 kDa allergen with homology to chitin-binding proteins, is immunologically distinct from Der p 15, 18, and 23. It is located in the peritrophic membrane of fecal pellets. Der p 37 reacted with IgE antibodies from a third of HDM-allergic patients and induced specific basophil- and CD4+ T-cell activation. Der p 37 IgE-positive patients had significantly higher IgE levels to major HDM allergens, reacted with more HDM allergens, and had a higher risk (odds ratio = 3.1) of asthma compared to Der p 37-negative patients. CONCLUSIONS: Der p 37, a new Dp allergen recognized by a third of HDM-allergic patients, may serve as a surrogate marker for severe HDM sensitization and asthma.

[Impact of patient testimony in the education of medicine and pharmacy students]. / Patients acteurs et formation en oncologie pour futurs médecins et pharmaciens.

The initiative from patients suffering from cancer or having had the disease in speaking about their illness to medical students during an internship in a cancer control center of the faculty of medicine and to pharmacy students at the university of Strasbourg was implemented in 2014. This action was coordinated by the French Cancer League as part of the National French cancer plan 3. After training, ten patients teachers were able to freely and spontaneously explain their diagnostic and therapeutic journey as well as their feelings about the disease and their relationship with their oncologists in front of 187 medical students and 131 pharmacy students. A moderator, often a former cancer teacher, helped coordinate the discussions. Questionnaires were given to students, patients teachers and moderators in order to assess the merits of the action and the expected benefits at the end of the training. A second questionnaire was sent to the students six months after the interviews. The assessment was made by an independent firm. The students' responses were very favorable and this training met their expectations in almost 98% of the cases and 1/3 of the students were destabilized by this training. Patients teacher were very satisfied with their intervention and felt that they were able to convey a message. Six months later, the 30% of student respondents said that these testimonies had or could have an impact on their practices. This is the first assessment of the interest of resource patients in teaching cancer patients about medical and pharmacy students.

Macrophage functionality and homeostasis in response to oligoethyleneglycol-coated IONPs: Impact of a dendritic architecture.

The engineering of iron oxide nanoparticles (IONPs) for biomedical use has received great interest over the past decade. In the present study we investigated the biocompatibility of IONPs grafted with linear (2P) or generation 1 (2PG1) or 2 (2PG2) dendronized oligoethyleneglycol units in THP-1-derived macrophages. To evaluate IONP effects on cell functionality and homeostasis, mitochondrial function (MTT assay), membrane permeability (LDH release), inflammation (IL-8), oxidative stress (reduced glutathione, GSH), NLRP3 inflammasome activation (IL-1ß) and nanoparticle cellular uptake (intracellular iron content) were quantified after a 4-h or 24-h cell exposure to increasing IONP concentrations (0-300 µg Fe/mL). IONPs coated with a linear molecule, NP10COP@2P, were highly taken up by cells and induced significant dose-dependent IL-8 release, oxidative stress and NLRP3 inflammasome activation. In comparison, IONPs coated with dendrons of generation 1 (NP10COP@2PG1) and 2 (NP10COP@2PG2) exhibited better biocompatibility. Effect of the dendritic architecture of the surface coating was investigated in a kinetic experiment involving cell short-term exposure (30 min or 1 h 30) to the two dendronized IONPs. NP10COP@2PG2 disrupted cellular homeostasis (LDH release, IL-1ß and IL-8 secretion) to a greater extend than NP10COP@2PG1, which makes this last IONP the best candidate as MRI contrast or theranostic agent.

How In Vitro Assays Contribute to Allergy Diagnosis.

Diagnosis of allergic disorders is based upon the clinical history of the disease, the immunoglobulin E (IgE) antibody response, and the allergen exposure. During the last decade, many changes have occurred in the in vitro diagnostic tests used in daily practice. The most important one is the use of allergenic molecules, which helps to define severe profile of allergy and/or to better understand cross-reactivity. The correlation between IgE sensitization and bronchial or nasal response in provocation tests is not so clear, which implies that such tests are still helpful in allergy diagnosis. In order to strengthen the link between a real allergen exposure and allergic symptoms, environmental allergen load assessment can be performed. For clinicians, it appears obvious to know the pollen count to treat their patients; however, they rarely measure the allergen load in the indoor environment, while nowadays home-tests (semi-quantitative or quantitative) make the assessment very easy. In the future, assessment of the environmental exposure (preferably with an indoor technician) of an allergic patient should take into account not only the allergens but also the other indoor pollutants, which could enhance respiratory symptoms in allergic patients.

Cationic DOPC-Detergent Conjugates for Safe and Efficient in Vitro and in Vivo Nucleic Acid Delivery.

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC-C12 E4 conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C12 E4 through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C12 E4 produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic-lipid-mediated gene transfer in animals.

DOPC-Detergent Conjugates: Fusogenic Carriers for Improved In Vitro and In Vivo Gene Delivery.

Phospholipid-detergent conjugates are proposed as fusogenic carriers for gene delivery. Eleven compounds are prepared and their properties are investigated. The ability of the conjugates to promote fusion with a negatively charged model membrane is determined. Their DNA delivery efficiency and cytotoxicity are assessed in vitro. Lipoplexes are administered in the mouse lung, and transgene expression Indeterminate inflammatory activity are measured. The results show that conjugation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with C12 E4 produces a carrier that can efficiently deliver DNA to cells, with negligible -associated toxicity. Fusogenicity of the conjugates shows good correlation with in vitro transfection efficiency and crucially depends on the length of the polyether moiety of the detergent. Finally, DOPC-C12 E4 reveals highly potent for in vivo DNA delivery and favorably compares to GL67A, the current golden standard for gene delivery to the airway, opening the way for further promising developments.

Asthma management and inhalation techniques among community pharmacists in 2009: a comparison with the 1999 survey.

OBJECTIVE: In a 1999 survey, community pharmacists from the Alsace region of France had a reasonably good knowledge of asthma treatment and prevention, but their skill in the use of asthma inhalation devices left room for improvement. Since then, health authorities have encouraged the involvement of community pharmacists in patient care and education in order to improve asthma control. The aim of this study was to assess the change in the knowledge of asthma management and inhaler technique skills of community pharmacists in the same geographic area after a 10-year interval. METHODS: In 2009, 86 randomly selected community pharmacists from the Alsace region answered a standardized questionnaire about their theoretical knowledge of and practical attitude toward asthma management and inhaled delivery systems, following which their skills in the use of four inhalation devices (pressurized metered-dose inhaler (pMDI) with/without a spacer, breath-actuated pMDI and dry powder inhaler (DPI)) were evaluated. RESULTS: Very few pharmacists were required to manage an acute asthma exacerbation at the pharmacy, but all responded well by administering a short-acting inhaled ß2-agonist. Theoretical knowledge of asthma management (criteria of severity of asthma exacerbation, guidelines and drugs triggering asthma exacerbations) was still average. Compared with 1999, they were twice as confident in demonstrating inhaler use, and their skills in using the pMDI, breath-actuated pMDI and DPI had improved significantly (p < 0.001). CONCLUSIONS: Since 1999, pharmacists' skill in the use of inhalers has improved, but theoretical knowledge of asthma management is still average, pointing to the importance of continuing pharmaceutical education.

Exposure to multi-walled carbon nanotubes results in aggravation of airway inflammation and remodeling and in increased production of epithelium-derived innate cytokines in a mouse model of asthma.

With the development of nanotechnologies, the potential adverse effects of nanomaterials such as multi-walled carbon nanotubes (MWCNT) on the respiratory tract of asthmatics are questioned. Furthermore, investigations are necessary to understand how these effects might arise. In the present study, we hypothesized that epithelium-derived innate cytokines that are considered as important promoting factors in allergy may contribute to an aggravating effect of MWCNT on asthma. We investigated in the mouse the effect of MWCNT on systemic immune response and airway inflammation and remodeling induced by the most frequent allergen so far associated with asthma, house dust mite (HDM), and we examined the production of the innate cytokines thymic stromal lymphopoietin (TSLP), IL-25, IL-33, and GM-CSF. Mice exposed to HDM exhibited specific IgG1 in serum and inflammatory cell infiltration, and increased Th2 cytokine production, mucus hyperproduction, and collagen deposition in the airways when compared to naïve animals. Levels of total IgG1 and HDM-specific IgG1, influx of macrophages, eosinophils and neutrophils, production of collagen, TGF-ß1, and mucus, as well as levels of IL-13, eotaxin, and TARC, were dose-dependently increased in mice exposed to HDM and MWCNT compared to HDM alone. These effects were associated with an increased production of TSLP, IL-25, IL-33, and GM-CSF in the airways. Our data demonstrate that MWCNT increase in a dose-dependent manner systemic immune response, as well as airway allergic inflammation and remodeling induced by HDM in the mouse. Our data suggest also a role for airway epithelium and innate cytokines in these effects.

Identification of Der p 23, a peritrophin-like protein, as a new major Dermatophagoides pteronyssinus allergen associated with the peritrophic matrix of mite fecal pellets.

The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.

Immunomodulatory properties of multi-walled carbon nanotubes in peripheral blood mononuclear cells from healthy subjects and allergic patients.

In the present study, we investigated the immunomodulatory activity of multi-walled carbon nanotubes (MWCNTs) in peripheral blood mononuclear cells (PBMCs) from healthy donors and mite-allergic subjects. Freshly prepared PBMCs, stimulated or not with Toll-like receptor (TLR)1-9 agonists, a T cell mitogen (phytohemagglutinin A) or mite allergen extract were cultured in the presence or absence of MWCNTs. Secretion of TNF-α, IL-2, IL-5, IL-6, IL-12/23p40 or IFN-γ was quantified in the culture supernatants by ELISA. Basal secretion of all the cytokines was not altered by MWCNTs in PBMCs from both healthy donors and allergic subjects. In PBMCs from healthy donors, TNF-α, IL-6 and IL-12/23p40 secretion in response to the TLR4 agonist, lipopolysaccharide was however increased in a dose-dependent manner by MWCNTs. Significant increases in the release of these cytokines were also observed in PBMCs stimulated with a TLR2 or TLR3 agonist. MWCNTs also increased the release of IL-2 and IFN-γ by PBMCs stimulated with a T cell mitogen. In contrast, MWCNTs inhibited allergen-induced IL-5 secretion by PBMCs from mite-allergic subjects. As well, MWCNTs altered the capacity of PBMC-derived monocytes to differentiate into functional dendritic cells. All together, our data suggest that according to its immune cell target, MWCNTs may either promote or suppress immune responses in humans. Further investigations are necessary to fully understand the complexity behind interactions of engineered nanoparticles with the immune system.

A dynamic system for single and repeated exposure of airway epithelial cells to gaseous pollutants.

In vitro models are promising approaches to investigate the adverse effects and the mode of action of air pollutants on the respiratory tract. We designed a dynamic system that allows the single or repeated exposure of cultured cells to two major indoor air gaseous pollutants, formaldehyde (HCHO) and nitrogen dioxide (NO2), alone or as a mixture. In this system, the Calu-3 human bronchial epithelial cell line was exposed at the air-liquid interface (ALI) or submerged by culture medium to synthetic air or to target concentrations of HCHO and/or NO2 once or on 4 consecutive days before assessment of cell viability and necrosis, IL-6 and IL-8 release and trans-epithelial electrical resistance. Our data showed that whereas the ALI method can be used for single short-term exposures only, the submerged method provides the possibility to expose Calu-3 cells in a repeated manner. As well, we found that repeated exposures of the cells to HCHO and NO2 at concentrations that can be found indoors triggered a significant decrease in cell metabolism and an increase in IL-8 release that were not evoked by a single exposure. Thus, our work highlights the fact that the development of systems and methods that allow repeated exposures of cultured cells to gaseous compounds in mixtures is of major interest to evaluate the impact of air pollution on the respiratory tract.

Varying allergen composition and content affects the in vivo allergenic activity of commercial Dermatophagoides pteronyssinus extracts.

BACKGROUND: Diagnosis and immunotherapy of house-dust mite (HDM) allergy is still based on natural allergen extracts. The aim of this study was to analyze commercially available Dermatophagoides pteronyssinus extracts from different manufacturers regarding allergen composition and content and whether variations may affect their allergenic activity. METHODS: Antibodies specific for several D. pteronyssinus allergens (Der p 1, 2, 5, 7, 10 and 21) were used to analyze extracts from 10 different manufacturers by immunoblotting. Sandwich ELISAs were used to quantify Der p 1 and Der p 2 in the extracts. Mite-allergic patients (n = 45) were skin-tested with the extracts and tested for immunoglobulin E (IgE) reactivity to a panel of 10 mite allergens (Der p 1, 2, 4, 5, 7, 8, 10, 14, 20 and 21) by dot blot. RESULTS: Only Der p 1 and Der p 2 were detected in all extracts but their concentrations and ratios showed high variability (Der p 1: 6.0-40.8 µg ml(-1); Der p 2: 1.7-45.0 µg ml(-1)). At least 1 out of 4 allergens (i.e. Der p 5, 7, 10 and 21) was not detected in 8 of the studied extracts. Mite-allergic subjects showed different IgE reactivity profiles to the individual mite allergens, the extracts showed different allergenic activity in skin-prick tests and false-negative results. CONCLUSIONS: Commercially available D. pteronyssinus extracts lack important allergens, show great variability regarding allergen composition and content and some gave false-negative diagnostic test results in certain patients.

Formaldehyde interferes with airway epithelium integrity and functions in a dose- and time-dependent manner.

Formaldehyde (HCHO) is a common indoor air pollutant. To assess its potential role and mechanism of action in asthma, we exposed the bronchial epithelial cell lines Calu-3 and 16HBE to HCHO (70-7000 µM) according to two exposure schedules (30 min and 24 h), before measuring cell viability, necrosis and apoptosis, reactive oxygen species production, cytokine release, as well as trans-epithelial electrical resistance (TEER) of cell monolayers. Whereas exposure to HCHO for 30 min had a limited effect on cell viability, exposure for 24h to 1400-7000 µM HCHO induced a pronounced dose-dependent cell death. The important decrease in cell viability observed after 24h exposure to the highest concentrations of HCHO (1400-7000 µM) was accompanied by important LDH release and ROS production, whereas a 4h exposure to lower HCHO concentrations (350 µM) induced cell apoptosis. Also, exposure to HCHO for 30 min dose-dependently inhibited basal and lipopolysaccharide-induced interleukin-6 (IL-6) and IL-8 production by bronchial epithelial cells. As well, HCHO triggered a dose- and time-dependent decrease in TEER of Calu-3 cell monolayers. The present work demonstrates that HCHO interferes with airway epithelium integrity and functions, and may thus modulate the onset and the severity of asthma. However, importantly, conditions of exposure to HCHO, e.g. level and duration, are determinant in the nature of the effects triggered by the pollutant.

Bronchial challenge test in asthmatics sensitized to mites: role of particle size in bronchial response.

Although major house dust mite allergen (Der p 1) is carried mainly on large particles (>10 microm), standard bronchial challenge tests (BCT) use nebulizers that deliver smaller particles (sizes from 1 to 5 microm) and may therefore not reflect actual domestic exposure. The objective of this study was to evaluate the influence of particle size of Dermatophagoides pteronyssinus extract on bronchial response. Specific BCT were performed with different mass median aerodynamic diameters (MMAD): 1.1, 5.6, and 9.7 microm. Each of the 19 mite-sensitized patients underwent mite BCT three times, once with each nebulizer. IL-5 levels were assessed in induced sputum and blood samples. The PD(20) for Der p 1 differed substantially with particle size, with less Der p 1 (11.2 ng) needed to produce a PD(20) with the largest particles (9.7 microm), compared to 18.1 ng for the 5.6 microm particles and 142.5 ng for the 1.1 microm particles (p < 0.0001). Large particles also induced an early phase response significantly more often than small particles (100% vs. 63%). Although the late phase reaction (LPR) frequency was similar with all three particle sizes, lower mean oral corticosteroid doses were needed to treat LPR with the largest particles (23 mg), compared to the smaller particles, with 34 mg for the 5.6 microm particles and 51 mg for the 1.1 microm. The 1.1 microm particles produced a significantly greater increase in IL-5 concentrations in sputum and blood compared to the larger particles. Large particles clearly play a role in the immediate bronchial response in asthmatics sensitized to mites and, therefore, should be included in pharmacological studies in humans.

[The role of the environment in asthma: hypotheses and contradictions]. / Le Rôle de l'enironnement dans l'asthme: hypothèses et contradictions.

Three hypotheses are described to explain the relation between allergens and environmental co-factors and the onset of atopy: the hygiene hypothesis, the allergenic hypothesis, and the high exposure tolerance inducing a Th2 derived response with blocking IgG4 synthesis. None of these hypotheses have been confirmed. It seems thus difficult to give recommendations for primary prevention of allergic diseases until results of prospective studies allow to consider a more precise behaviour. In contrast, subjects sensitized and exposed to allergens present an increased risk to develop asthma or non specific bronchial hyperreactivity. Therefore, secondary prevention appears as an essential method for treatment of allergic disease, with clinical benefits on symptoms which have recently been demonstrated in a clinical study.

Benzo(a)pyrene-coated onto Fe2O3 particles-induced apoptotic events in the lungs of Sprague-Dawley rats.

Because benzo(a)pyrene (B(a)P)-coated onto hematite (Fe(2)O(3)) particle-induced adverse effects might alter cell homeostasis in lungs, we investigated the induction of some apoptotic events by such a concurrent exposure on this relevant organ target. Sprague-Dawley rats were intratracheally instilled with Fe(2)O(3) (3 mg), B(a)P (3 mg) or B(a)P (3 mg)-coated onto Fe(2)O(3) particles (3 mg). Forty-eight hours later, both the tumor necrosis factor-receptor and the mitochondrial pathways were studied. We found that exposure to B(a)P (1.13-fold, P<0.05) or to B(a)P-coated onto Fe(2)O(3) particles (1.15-fold, P<0.05) increased caspase 3 activity. However, only the concurrent exposure activated both the caspases 8 (1.21-fold, P<0.05) and 9 (1.27-fold, P<0.05). After exposure to either chemical alone, there was a discrepancy between the findings on tumor necrosis factor-alpha and caspase 8, on one hand, and on cytochrome c and caspase 9, on the other hand. Hence, we suggested that the oxidative stress induced by Fe(2)O(3) or B(a)P will continuously lower or deplete caspase activities, thereby reducing or even avoiding the activation of the apoptotic pathways. In addition, transcriptional induction of p53 gene by Fe(2)O(3) (1.73-fold, P<0.01) or B(a)P-coated onto Fe(2)O(3) particles (1.53-fold, P<0.01) was observed. Taken together, the present results support the underlying hypothesis that the influence of Fe(2)O(3) in B(a)P/Fe(2)O(3) mixtures on the ability of B(a)P to induce some of the events firmly involved in the apoptotic pathways will also be one of the ways that Fe(2)O(3) can affect B(a)P toxicity in lungs.