Nasal Instillation Research Articles

Abstract B23: Pushing Myc inhibition towards the clinic by direct delivery of cell-penetrating peptides

Abstract Inhibiting Myc has long been regarded as a promising cancer treatment. However, clinical Myc inhibition was considered unfeasible due to its central role in normal proliferation and the difficulties of targeting a nuclear transcription factor. The expression of Omomyc (a Myc inhibitor derived from the dimerization and DNA-binding domain of Myc) in the KRasG12D non-small cell lung cancer (NSCLC) mouse model challenged these assumptions, as it resulted in dramatic tumor clearance with only limited and well tolerated side effects in normal tissues (Soucek et al., 2008 and 2013). Omomyc expression proved equally potent in several other mouse models of cancer, revealing the huge potential of this inhibitory approach against multiple cancer types including papilloma, pancreas and glioma (Soucek et al., 2004; Sodir et al., 2011; Annibali et al., 2014). Recently, Max*, a b-HLH-LZ peptide derived from Myc's obligate protein partner Max, was shown to spontaneously enter cells (Montagne et al., 2012). As Omomyc and Max* display high structural homology, we hypothesized that Omomyc could also behave as a cell-penetrating peptide and thus recapitulate the effects of its transgenic counterpart. Our preliminary results show that the Omomyc peptide is well folded in solution; it transduces into cancer cells and effectively stops their proliferation in a dose-dependent manner.In vivo, nasal instillation of fluorescently-labeled Omomyc peptide leads to its rapid distribution to lungs and brain, as well as to other organs (G.I. tract, liver), as observed by IVIS® imaging and immunohistochemistry. Finally, a short treatment with the Omomyc peptide reduces the tumor size and number of Ki67 positive cells in the KRasG12D-induced NSCLC mouse model. In summary, the Omomyc cell penetrating peptide represents a new opportunity to pharmacologically inhibit Myc in a variety of malignant diseases. Citation Format: Marie-Eve Beaulieu, Toni Jauset, Daniel Massó-Valles, Jonathan R. Whitfield, Erika Serrano del Pozo, Cynthia Tremblay, Loïka Maltais, Martin Montagne, Pierre Lavigne, Laura Soucek. Pushing Myc inhibition towards the clinic by direct delivery of cell-penetrating peptides. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B23.

WS01.1 rAAV2/5 encoding a truncated CFTR rescues the CF phenotype in intestinal organoids and a CF mouse model

To date, the majority of CF patients receive symptomatic treatment. Gene therapy offers the potential to cure CF in mutation-independent manner. Here, we evaluate rAAV2/5 gene therapy for CF in human intestinal organoids and a ΔF508 mouse model. First, we studied the longevity of gene expression after a single administration of a rAAV2/5-Fluc to mouse airways. This resulted in relatively stable gene expression up to 15 months with only a 4-fold decrease in bioluminescent signal in lungs. Due to the rAAV size limit, we used a truncated CFTR missing a portion of the R-domain, CFTRΔR (Ostedgaard et al., PNAS, 2005). First, we evaluated its functionality in a physiological relevant and highly translational organoid model. These data demonstrate that the therapeutic vector rAAV-CFTRΔR is functional and that the ion transport-induced organoid swelling is CFTR-specific. Indeed, despite the expression cassette being at the limit of the rAAV packaging capacity, intact genomes are incorporated into vector particles, that allow efficient second strand DNA synthesis and transgene expression. Finally, we administered rAAV2/5-CFTRΔR to ΔF508 mice by nasal instillation. 2–4 weeks later, we demonstrated a response to low-chloride and forskolin perfusion in 6 out of 8 mice by nasal potential differences, indicating restoration of chloride transport across nasal mucosa. In conclusion, we obtained sustained reporter gene expression in murine airways and demonstrated restoration of the CF phenotype in CF organoids and a CF mouse model. Our results underscore the therapeutic potential of rAAV2/5-CFTRΔR for CF gene therapy opening new avenues towards a definitive cure for all CF patients.

370. F/HN Pseudotyped Lentivirus Generates Therapeutically Relevant and Long-Lasting Alpha-1-Antitrypsin Expression in Mouse Lung

A protease/anti-protease imbalance is a characteristic feature of inflammatory lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). However, alpha-1-antitrypsin (AAT) enzyme replacement therapy (ERT) trials have not shown conclusive evidence of a therapeutic benefit. This may be due to inefficient protein delivery to the lung and/or the half-life of the protein. The UK Cystic Fibrosis Gene Therapy Consortium (GTC) has pseudotyped a simian immunodeficiency viral vector with the Sendai virus F and HN proteins (rSIV.F/HN) and selected an internal synthetic promoter/enhancer consisting of the elongation factor 1α promoter and the CMV enhancer (hCEF) for efficient transduction of airway epithelial cells. Here, we assessed whether transduction of murine lungs with rSIV.F/HN with a human AAT transgene generates therapeutic levels of AAT. Mice were transduced with rSIV.F/HN-hCEF-AAT (2e8 TU/mouse) by nasal instillation and culled 10 days after transduction. AAT levels in lung homogenate and broncho-alveolar lavage fluid (BALF) were at least 3 logs above controls (p<0.05). A urea assay was used to determine that lavage diluted the epithelial lining fluid (ELF) by ≈40-fold, which allowed us to calculate the AAT concentration in ELF to be 92±28 mg/ml (= 1.4 μμ). In the context of AAT deficiency, an ELF concentration of 70 mg/ml (1.1 μM) AAT is of therapeutic relevance. For comparison transfection of mouse lung with the cationic lipid GL67A complexed to pCMV-AAT only led to 0.4±0.1 mg/ml (n=6) of AAT in ELF. A neutrophil elastase (NE) activity assay showed that the recombinant AAT successfully neutralised NE activity (p<0.05). In a separate experiment, mice were treated with rSIV.F/HN-hCEF-AAT (4e7 TU/mouse) and quantification of AAT 7 and 90 days post-transduction showed that expression was stable over this period. We also transduced mice with rSIV.F/HN-hCEF carrying a secreted Gaussia luciferase reporter gene (1e7 TU/mouse) and showed that stable expression in lung and BALF persisted for at least 12 months. Here, we also demonstrate for the first time that rSIV.F/HN transduction of lung generates significant (p<0.05) levels of GLux and AAT recombinant proteins in serum. AAT enzyme replacement therapy is currently being assessed for the treatment of a range of diseases including diabetes and graft-vs-host disease, but shortage of human plasma-derived protein, as well as high costs, may limit the application. In conclusion, rSIV.F/HN produces therapeutically relevant and long lasting levels of AAT in murine lung. In addition, we showed that AAT escapes from the lung into the circulation, suggesting that gene therapy may help to overcome some of the current bottlenecks of ERT.

171. Production of Therapeutically Relevant Levels of FVIII After Transduction of Lungs With F/HN-Pseudotyped Lentivirus

We have previously shown that the lung can be used as “factory” for production of secreted proteins, which are released into the circulation, using Sendai virus-mediated gene transfer. However, gene expression was transient and repeated administration of the vector was not feasible due to induction of immune responses. We have developed a lentiviral vector which is specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV.F/HN) to allow efficient transduction of the airways. The unique features of rSIV.F/HN, namely stable expression for >20 months after a single dose and efficient transduction after repeated administration, make the vector an attractive candidate for a large range of disease indications. Here, we first transduced mouse lung with rSIV.F/HN carrying the secreted reporter gene Gaussia luciferase (GLux) or a control virus by nasal instillation (1e6 transduction units (TU)/mouse, n=5-6/group). Persistent levels of GLux expression were detectable in lung (3 logs above control) and broncho-alveolar lavage fluid (BALF, 4 logs above control) for at least 12 months. Importantly, even this modest dose of virus lead to significant (p<0.01) levels of GLux in serum (274±72 RLU/ul, control: 41±6 RLU/ul) which persisted for at least 12 months further supporting the hypothesis that the lung is a suitable, non-invasive factory for production of secreted proteins. Gene therapy strategies for haemophilia have focussed on intravenous or intramuscular delivery of the gene transfer agent. Here, we treated the murine lung with rSIV.F/HN carrying the FVIII cDNA (1.6e8-3.4e8 TU/mouse, n=3-4/group) or placebo and assessed whether therapeutically relevant levels of FVIII can be produced. Significant (p<0.05) and dose-related levels of FVIII were detectable in lungs and BALF 10 and 28 days post-transduction. Dose-related levels of FVIII were also detectable in plasma, which reached a therapeutically relevant level of 3.0% of normal 1 month after gene transfer. These data support the concept that rSIV.F/HN-mediated transduction of lungs can produce therapeutically relevant and persistent levels of recombinant protein in blood.

CXCR1/CXCR2 antagonist CXCL8(3-74)K11R/G31P blocks lung inflammation in swine barn dust-instilled mice

Inhalation of agricultural occupational dusts from swine confinement facilities can result in lung inflammation. The innate immune response to organic barn dusts results in production of a number of pro-inflammatory factors in the lungs of barn workers such as cytokines, chemokines, and an influx of neutrophils. Many of these inflammatory factors are influenced by the chemokine CXCL8/IL-8 (KC or MIP-2 in mice). Previously, we have demonstrated that an endotoxin-independent component of swine barn dust extract (SBE) elevates lung chemokines in a protein kinase C (PKC)-dependent manner resulting in the significant formation of lung inflammatory cell infiltrates in a mouse model of SBE injury. In this study we test the ability of a CXCR1/CXCR2 antagonist, CXCL8(3-74)K11R/G31P (G31P) to block many of the features of lung-inflammation in response to challenge with SBE in an established mouse exposure system. Injection of G31P concurrent with SBE nasal instillation over a course of 3 weeks significantly reduced neutrophil accumulation in the lungs of barn dust exposed animals compared to those given SBE alone. There was a similar reduction in pro-inflammatory cytokines and chemokines IL-6, KC, and MIP-2 in SBE plus G31P-treated mice. In addition to excreted products, the receptors ICAM-1, CXCR1, and CXCR2, which all were elevated with SBE exposure, were also decreased with G31P treatment. SBE activation of PKCα and PKCε was reduced as well with G31P treatment. Thus, G31P was found to be highly effective at reducing several features of lung inflammation in mice exposed to barn dust extracts.

Chronic exposure of diesel exhaust particles induces alveolar enlargement in mice.

BackgroundDiesel exhaust particles (DEPs) are deposited into the respiratory tract and are thought to be a risk factor for the development of diseases of the respiratory system. In healthy individuals, the timing and mechanisms of respiratory tract injuries caused by chronic exposure to air pollution remain to be clarified.MethodsWe evaluated the effects of chronic exposure to DEP at doses below those found in a typical bus corridor in Sao Paulo (150 μg/m3). Male BALB/c mice were divided into mice receiving a nasal instillation: saline (saline; n = 30) and 30 μg/10 μL of DEP (DEP; n = 30). Nasal instillations were performed five days a week, over a period of 90 days. Bronchoalveolar lavage (BAL) was performed, and the concentrations of interleukin (IL)-4, IL-10, IL-13 and interferon-gamma (INF-γ) were determined by ELISA-immunoassay. Assessment of respiratory mechanics was performed. The gene expression of Muc5ac in lung was evaluated by RT-PCR. The presence of IL-13, MAC2+ macrophages, CD3+, CD4+, CD8+ T cells and CD20+ B cells in tissues was analysed by immunohistochemistry. Bronchial thickness and the collagen/elastic fibers density were evaluated by morphometry. We measured the mean linear intercept (Lm), a measure of alveolar distension, and the mean airspace diameter (D0) and statistical distribution (D2).ResultsDEP decreased IFN-γ levels in BAL (p = 0.03), but did not significantly alter IL-4, IL-10 and IL-13 levels. MAC2+ macrophage, CD4+ T cell and CD20+ B cell numbers were not altered; however, numbers of CD3+ T cells (p ≤ 0.001) and CD8+ T cells (p ≤ 0.001) increased in the parenchyma. Although IL-13 (p = 0.008) expression decreased in the bronchiolar epithelium, Muc5ac gene expression was not altered in the lung of DEP-exposed animals. Although respiratory mechanics, elastic and collagen density were not modified, the mean linear intercept (Lm) was increased in the DEP-exposed animals (p ≤ 0.001), and the index D2 was statistically different (p = 0.038) from the control animals.ConclusionOur data suggest that nasal instillation of low doses of DEP over a period of 90 days results in alveolar enlargement in the pulmonary parenchyma of healthy mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0172-z) contains supplementary material, which is available to authorized users.

The anterior commissure is a pathway for contralateral spread of herpes simplex virus type 1 after olfactory tract infection

Herpes simplex encephalitis (HSE), targeting the limbic system, is the most common cause of viral encephalitis in the Western world. Two pathways for viral entry to the central nervous system (CNS) in HSE have been suggested: either via the trigeminal nerve or via the olfactory tract. This question remains unsettled, and studies of viral spread between the two brain hemispheres are scarce. Here, we investigated the olfactory infection as a model of infection and tropism of herpes simplex virus 1 (HSV-1), the causative agent of HSE, in the CNS of rats. Rats were instilled with HSV-1 in the right nostril and sacrificed 1-6days post-infection, and tissues were analysed for viral spread using immunohistochemistry and quantitative PCR (qPCR). After nasal instillation, HSV-1 infected mitral cells of the olfactory bulb (OB) on the right side only, followed by limbic encephalitis. As a novel finding, the anterior commissure (AC) conveyed a rapid transmission of virus between the right and the left OB, acting as a shortcut also between the olfactory cortices. The neuronal cell population that conveyed the viral infection via the AC was positive for the water channel protein aquaporin 9 (AQP9) by immunohistochemistry. Quantification of AQP9 in cerebrospinal fluid samples of HSE patients showed increment as compared to controls. We conclude that the olfactory route and the AC are important for the spread of HSV-1 within the olfactory/limbic system of rats and furthermore, we suggest that AQP9 is involved in viral tropism and pathogenesis of HSE.

Novel dendrimeric peptide: Immune response and protection against Foot-and-mouth disease virus (FMDV) induced by synthetic peptides in cattle

Novel dendrimeric peptide: Immune response and protection against Foot-and-mouth disease virus (FMDV) induced by synthetic peptides in cattle

Persistence of asthmatic response after ammonium persulfate-induced occupational asthma in mice.

IntroductionSince persulfate salts are an important cause of occupational asthma (OA), we aimed to study the persistence of respiratory symptoms after a single exposure to ammonium persulfate (AP) in AP-sensitized mice.Material and MethodsBALB/c mice received dermal applications of AP or dimethylsulfoxide (DMSO) on days 1 and 8. On day 15, they received a single nasal instillation of AP or saline. Airway hyperresponsiveness (AHR) was assessed using methacholine provocation, while pulmonary inflammation was evaluated in bronchoalveolar lavage (BAL), and total serum immunoglobulin E (IgE), IgG1 and IgG2a were measured in blood at 1, 4, 8, 24 hours and 4, 8, 15 days after the single exposure to the causal agent. Histological studies of lungs were assessed.ResultsAP-treated mice showed a sustained increase in AHR, lasting up to 4 days after the challenge. There was a significant increase in the percentage of neutrophils 8 hours after the challenge, which persisted for 24 hours in AP-treated mice. The extent of airway inflammation was also seen in the histological analysis of the lungs from challenged mice. Slight increases in total serum IgE 4 days after the challenge were found, while IgG gradually increased further 4 to 15 days after the AP challenge in AP-sensitized mice.ConclusionsIn AP-sensitized mice, an Ig-independent response is induced after AP challenge. AHR appears immediately, but airway neutrophil inflammation appears later. This response decreases in time; at early stages only respiratory and inflammatory responses decrease, but later on immunological response decreases as well.

Changes of serum parameters of TiO2 nanoparticle-induced atherosclerosis in mice

The evaluation of toxicological effects of nanoparticulate matter is increasingly important due to their growing occupational use and presence as compounds in consumer products. Numerous studies have shown that exposure to nanosized particles lead to systemic inflammation in experimental animals, but whether long-term exposure to nanosized particles induces atherogenesis is rarely evaluated. In the current study, mice were continuously exposed to TiO2 nanoparticles (NPs) at 1.25, 2.5, or 5mg/kg body weight, administered by nasal instillation for nine consecutive months, and the association between serum parameter changes and atherosclerosis in mice were investigated. The present findings suggested that chronic exposure to TiO2 NPs resulted in atherogenesis coupling with pulmonary inflammation, increased levels of serum triglycerides, glucose, total cholesterol, low-density lipoprotein cholesterol, advanced glycation end products, reactive oxygen species, NAD(P)H oxidases 4, C-reaction protein, E-selectin, endothelin-1, tissue factor, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, and reduced levels of serum high-density lipoprotein cholesterol, nitric oxide and tissue plasminogen activator. Our study suggests an association of long-term exposure to TiO2 NPs with atherosclerosis and pulmonary inflammation. This finding demonstrates the hypothesized role of TiO2 NPs as a risk factor for atherogenesis.

Manganese-enhanced magnetic resonance imaging for studying olfactory dysfunction in rats

To study the olfactory function in rats by manganese-enhanced magnetic resonance imaging (MEMRI) and explore the regeneration of olfactory system from the imaging. Thirty-five adult male Sprague-Dawley (SD) rats were randomly divided into three groups. Twenty rats with bilateral nasal instillation of TritonX-100 were used as olfactory dysfunction model group (M group). The rats in this group received menthocamphorate stimulation. Ten rats with bilateral nasal instillation of sterile saline were used as olfactory normal group (N group), and were randomly divided into two groups:one group received menthocamphorate stimulation (N1 group), another group received odorless air (N2 group). The remaining five rats were used as the blank control (control group). All images were acquired with a 7.0 T micro-MR scanner. Signal-to-noise ratios (SNR) in the olfactory bulb (OB) were measured by Image J. MEMRI could clearly show the normal olfactory pathway in rats. MEMRI displayed a reversible change during the stages of olfactory recovery after injury. For the olfactory dysfunction model group (M group), the total volume of rat olfactory bulb at the initial, the 10th day, the 20th day, the 30th day and the 60th day were (49.44 ± 0.81), (32.85 ± 0.79), (27.78 ± 1.07), (35.89 ± 1.04), (43.63 ± 1.13) mm(3) respectively. At the 20th day after olfactory injury, the SNR in the OB was the lowest for 9.78 ± 0.07, when at the 60th day, the SNR recovered to 30.68 ± 1.01, which increased to near normal (N1group, 33.08 ± 0.15; N2 group, 31.31 ± 1.12), the SNR had no significant difference among the three groups (F = 3.04, P > 0.05). The MEMRI is an objective method to detect the olfactory function, and the olfactory system has the regenerative property after injury.

A treatment with a protease inhibitor recombinant from the cattle tick (Rhipicephalus Boophilus microplus) ameliorates emphysema in mice.

AimsTo determine whether a serine protease inhibitor treatment can prevent or minimize emphysema in mice.MethodsC57BL/6 mice were subjected to porcine pancreatic elastase (PPE) nasal instillation to induce emphysema and were treated with a serine protease inhibitor (rBmTI-A) before (Protocol 1) and after (Protocol 2) emphysema development. In both protocols, we evaluated lung function to evaluate the airway resistance (Raw), tissue damping (Gtis) and tissue elastance (Htis). The inflammatory profile was analyzed in the bronchoalveolar lavage (BALF) and through the use of morphometry; we measured the mean linear intercept (Lm) (to verify alveolar enlargement), the volume proportion of collagen and elastic fibers, and the numbers of macrophages and metalloprotease 12 (MMP-12) positive cells in the parenchyma. We showed that at both time points, even after the emphysema was established, the rBmTI-A treatment was sufficient to reverse the loss of elastic recoil measured by Htis, the alveolar enlargement and the increase in the total number of cells in the BALF, with a primary decrease in the number of macrophages. Although, the treatment did not control the increase in macrophages in the lung parenchyma, it was sufficient to decrease the number of positive cells for MMP-12 and reduce the volume of collagen fibers, which was increased in PPE groups. These findings attest to the importance of MMP-12 in PPE-induced emphysema and suggest that this metalloprotease could be an effective therapeutic target.

Oxidative stress and acute changes in murine brain tissues after nasal instillation of copper particles with different sizes.

We aim to investigate the biological effects of copper particles on the murine brain and their underlying mechanism after nasal instillation of copper particles. We choose different sizes and different concentrations of copper nanoparticles for mice intranasal use. Within one week, the mice were sacrificed. Pathological lesions of glial cells were detected by immunohistochemical assay. Immunohistochemical assay reveals that glial fibrillary acidic protein (GFAP) increased significantly in all experimental groups, especially in nanocopper groups. The ultrastructure of nerve cells was observed through TEM, whose results show that there were chromatin congregation and mitochondria shrinkage in the olfactory cells, and that there was increase of endoplasmic reticulum and disassociation of endoplasmic reticulum ribosomes in hippocampus, particularly in the nanocopper-groups. Oxidative stress indexes were determined with colorimetric methods. There was no significant increase in the antioxidative enzymes (GPX, GST, SOD) in brain tissues; however, significant increase of malondiadehyde (MDA) contents was only found in the Cu nanoparticle-exposed mice at the high dose of 40 mg per kg body weight. Based on the investigation into the biological effects of copper nanoparticles (23.5 nm) after intranasal instillation to the mice, we have found that copper particles can indeed enter into the olfactory bulb and then the deeper brain. The inhalation of high dose copper nanoparticles can induce severer lesions of brain in the experimental mice. The underlying mechanism of copper nanoparticles causing severe brain damage bears little connection with oxidative stress.

Intranasal exposure to uranium results in direct transfer to the brain along olfactory nerve bundles

Uranium olfactory uptake after intranasal exposure raises some concerns for people potentially exposed to airborne radionuclide contamination as the brain could be a direct target for these contaminants. A model of nasal instillation was used to elucidate the transport mechanisms of uranium to the brain and to map its localization. Increasing concentrations of depleted uranium containing solutions were instilled in the nasal cavity of adult male rats. Uranium concentrations were measured using inductively coupled plasma-mass spectrometry (ICP-MS) 4 h after instillation. Olfactory neuroepithelium cytoarchitecture was studied using immunohistochemistry experiments. Secondary ion mass spectrometry (SIMS) microscopy was performed to localize uranium in the olfactory system. ICP-MS analyses showed a frontal accumulation of uranium in the olfactory bulbs associated with a smaller increase in more caudal brain regions (frontal cortex, hippocampus and cerebellum). Uranium concentrations in the olfactory bulbs do not reach a saturation point. Olfactory nerve bundle integrity is not affected by uranium as revealed by immunohistochemistry. SIMS microscopy allowed us to show that uranium localization is mainly restricted to the olfactory neuroepithelium and around olfactory nerve bundles. It is subsequently detected in the olfactory nerve layer of the olfactory bulb. These results suggest the existence of a transcellular passage from the mucosa to the perineural space around axon bundles. Uranium bypasses the blood brain barrier and is conveyed to the brain via the cerebrospinal fluid along the olfactory nerve. Future studies might need to integrate this new contamination route to assess uranium neurotoxicity after nasal exposure.

Methionine oxidation in albumin by fine haze particulate matter: An in vitro and in vivo study

The potential effects of inhaled fine particulate matter (PM2.5), found in haze episodes, on the oxidation of the proteins in the lungs are not well understood. We investigated the effects of PM2.5 from haze episodes on protein oxidation. PM2.5 was collected from the air pollution in Beijing (BJ), Xian (XA), Xiamen (XM) and Hong Kong (HK) during a period of intensive haze episodes. The chemical characteristics of these samples and their effects on albumin oxidation were investigated. The levels of PM2.5 in BJ and XA were 4–6 times higher than in XM and HK. The concentrations of the polycyclic aromatic hydrocarbons (PAHs) components of the PM2.5 from BJ and XA were 10 times higher than those found in XM and HK. The haze PM2.5 increased oxidative stress. Addition of PM2.5 samples collected from haze episodes to albumin in vitro resulted in oxidation of methionine moieties; nasal instillation of PM2.5 suspensions in mice resulted in oxidation of methionine in the albumin in the bronchoalveolar lavage fluid. The methionine moieties participate in peptide chain crosslinking, and methionine oxidation in the albumin could be attributed to the PAH compounds. Our findings may be helpful in explaining the potential respiratory effects during haze episodes.

Shifted T-cell polarisation after agricultural dust exposure in mice and men

RationaleA low prevalence of asthma and atopy has been shown in farmers and agricultural workers. However, in these workers, a higher prevalence of respiratory symptoms has been reported, in which...

The Absence of CpG in Plasmid DNA–Chitosan Polyplexes Enhances Transfection Efficiencies and Reduces Inflammatory Responses in Murine Lungs

Chitosan polyplexes containing plasmid DNA (pDNA) have significant potential for pulmonary gene delivery applications. However, prior to using chitosan/pDNA polyplexes (CSpp) in clinical applications, their potential cytotoxicity needs to be investigated. In this study, we formulated 200–400 nm CSpp with amine to phosphate (N/P) ratios that ranged from 1 to 100. We compared two types of plasmids within CSpp: pDNA that was free of CpG sequences (CpG(−)) and pDNA that contained CpG sequences (CpG(+)). Both forms of CSpp showed low cytotoxicity when cultured with A549 and HEK293 cell lines in vitro. CSpp(CpG(−)) generated higher luciferase expression both in vitro, for A549 cells, and in vivo, compared with CSpp(CpG(+)). In addition, CSpp(CpG(−)) elicited milder inflammatory responses in mice one day subsequent to nasal instillation, as determined by proinflammatory cytokine levels within the bronchoalveolar lavage fluid. Our findings suggest that to achieve optimal gene expression with minimal cytotoxicity, inflammation, and oxidative stress, the N/P ratios and CpG sequences in the pDNA of CSpp need to be considered. These findings will inform the preclinical safety assessments of CSpp in pulmonary gene delivery systems.

A Quantitative Study of Airway Changes on Micro-CT in a Mouse Asthma Model: Comparison With Histopathological Findings

PurposeTo evaluate airway changes in ovalbumin-induced asthmatic mice in terms of postmortem micro-CT images and pathological findings.MethodsAsthma was induced in mice by intraperitoneal injection and nasal instillation of ovalbumin aluminium hydroxide into mice (experimental group, n=6), and another group of mice received intraperitoneal injection and nasal instillation of distilled phosphate-buffered saline (control group, n=6). Bronchial lumen area was measured in the main bronchial lumen of the distal third bronchial branch level (6 parts per each mouse) on axial scans of Micro-CT, using a Lucion's smart pen (semi-automated) and a curve pen (manual). Bronchial wall thickness was obtained in 4 sections (2 levels on either side) after the third bronchial branch by measuring the diameter which was perpendicular to the longitudinal axis of the main bronchus on curved Multi-planar reconstruction (MPR) images. Histologic slides were obtained from the lesion that was matched with its CT images, and bronchial wall thicknesses were determined.ResultsThe mean bronchial lumen area was 0.196±0.072 mm2 in the experimental group and 0.243±0.116 mm2 in the control group; the difference was significant. Bronchial wall thickness on micro-CT images (mean, 0.119±0.01 vs. 0.108±0.013 mm) and in pathological specimens (mean, 0.066±0.011 vs. 0.041±0.009 mm) were thicker in the experimental group than in the control group; bronchial wall thickness on micro-CT images correlated well with pathological thickness (for the experimental group, r=0.712; for the control group, r=0.46). The thick bronchial wall in the experimental group demonstrated submucosal hypertrophy along with goblet cell hyperplasia and smooth muscle hyperplasia.ConclusionsThe results of this study suggest that asthma may induce thickening of bronchial wall and narrowing of the lumen area on micro-CT images and that these results may significantly correlate with pathological findings.

The effectiveness of Ayurvedic oil based nasal instillation (Nasya) medicines for the treatment of facial paralysis (Ardita): a systematic review protocol

Review question/objective The objective of this review is to establish the effectiveness of oil based nasal instillation medicines in the treatment of Ardita (facial paralysis). The question that will be asked in the review is: Which of the commonly used Ayurvedic nasal instillation medicated oils is the most effective for treating Ardita either solely or in combination with other Ayurvedic medical interventions? Inclusion criteria Types of participants Adults (18-70 years of age) with Ardita (chronic or acute) are the participants to be included in this review. More specifically, the review will consider for inclusion studies that have examined the effectiveness of Naysa for treating acute or chronic Ardita in adults. An inclusive approach will be adopted with respect to geographical location of the participants with patients located in any country, and both rural and urban areas to be considered. Participants of any socio-economic status, both sexes and all ethnic origins will be considered. Studies whose participants have been pregnant women, adults older than 70 years and patients with allergic rhinitis, fever, intracranial tumour/haemorrhage and bilateral facial palsy will be excluded from the review. The reason for the lower age limit of 18 is because oil based Nasya is not usually administered to children, according to the ancient textual source Astanga Hridaya Sootra Sthanna. Types of intervention(s) and comparator(s) The review will include for consideration all quantitative studies conducted worldwide that have examined the effectiveness of nasal instillation of Ayurvedic oil-based herbal medicine. All studies that quantified the effectiveness of Nasya either administered by a therapist or by self administration in treating facial paralysis will be considered for inclusion. All dosages and frequencies of Nasya use will be considered and if possible how effectiveness varies with dosage and frequency of use will be detailed in the analysis. All studies in which the comparator was conventional medical management or placebo will be considered for inclusion. However, studies of Ayurvedic Nasya medicine in conjunction with conventional medicine, if any, will be excluded. As the objective is not only to shed light on the effectiveness of Nasya for treating facial paralysis, but also the effectiveness of one kind of Nasya medicine compared to another, all studies that have compared the effectiveness of one Nasya instillation medicine compared to another will be considered. Types of Outcomes The review will consider both Ayurvedic and Conventional medicine outcome assessment criterion as described in the research papers. With respect to the conventional medicine this will include, but not be limited to facial function as measured by the House-Brackmann grading of facial function measure. With respect to the Ayurvedic medicine approach this will include but not limited to Ayurvedic diagnostic scoring.

Isolation and Molecular Characterization of Two Lectins from Dwarf Elder (Sambucus ebulus L.) Blossoms Related to the Sam n1 Allergen

Sambucus species contain a number of lectins with and without antiribosomal activity. Here, we show that dwarf elder (Sambucus ebulus L.) blossoms express two d-galactose-binding lectins that were isolated and purified by affinity chromatography and gel filtration. These proteins, which we named ebulin blo (A-B toxin) and SELblo (B-B lectin)—blo from blossoms—were subjected to molecular characterization and analysis by MALDI-TOF mass spectrometry and tryptic peptide fingerprinting. Both lectins share a high degree of amino acid sequence homology with Sambucus lectins related to the Sam n1 allergen. Ebulin blo, but not SELblo, was highly toxic by nasal instillation to mice. Overall, our results suggested that both lectins would belong to an allergen family exemplified by Sam n1 and could trigger allergy responses. Furthermore, they raise a concern about ebulin blo toxicity.