Evaluation of Calu-3 cell lines as an in vitro model to study the inhalation toxicity of flavoring extracts.

This study aimed to evaluate the characteristics of Calu-3 cells as a model to examine the toxicological responses of inhalable substances. Calu-3 cells were grown to the confluence at an air-liquid interface (ALI) using a Transwell® permeable support system. The ALI resulted in biomimetic native bronchial epithelium displaying pseudostratified columnar epithelium with more microvilli and secretory vesicles. We further characterized and optimized the Calu-3 cell line model using ALI culturing conditions, immunolabeling of protein expression, ultrastructural analysis using scanning electron microscopy (SEM), and transepithelial electrical resistance (TEER) measurements, and then screened for the cytotoxicity of tobacco flavoring extracts. Calu-3 cells displayed dose-dependent responses when treated with the flavoring extract. Within 8-10 days, cell monolayers developed TEER ≥1000 Ω·cm2. During this time, Calu-3 cells exposed to flavoring extracts X01 and X06 exhibited a loss of cellular integrity and decreased ZO-1 and E-cadherin protein expression. In conclusion, we investigated the Calu-3 cell line culture conditions, culture time, and barrier integrity and tested the effect of six new synthetic tobacco flavoring extracts. Our data demonstrate that the Calu-3 human bronchial epithelial cell monolayer system is a potential in vitro model to assess the inhalation toxicity of inhalable substances.

Drug repurposing for COVID-19 based on an integrative meta-analysis of SARS-CoV-2 induced gene signature in human airway epithelium.

Drug repurposing has the potential to bring existing de-risked drugs for effective intervention in an ongoing pandemic-COVID-19 that has infected over 131 million, with 2.8 million people succumbing to the illness globally (as of April 04, 2021). We have used a novel `gene signature'-based drug repositioning strategy by applying widely accepted gene ranking algorithms to prioritize the FDA approved or under trial drugs. We mined publically available RNA sequencing (RNA-Seq) data using CLC Genomics Workbench 20 (QIAGEN) and identified 283 differentially expressed genes (FDR<0.05, log2FC>1) after a meta-analysis of three independent studies which were based on severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection in primary human airway epithelial cells. Ingenuity Pathway Analysis (IPA) revealed that SARS-CoV-2 activated key canonical pathways and gene networks that intricately regulate general anti-viral as well as specific inflammatory pathways. Drug database, extracted from the Metacore and IPA, identified 15 drug targets (with information on COVID-19 pathogenesis) with 46 existing drugs as potential-novel candidates for repurposing for COVID-19 treatment. We found 35 novel drugs that inhibit targets (ALPL, CXCL8, and IL6) already in clinical trials for COVID-19. Also, we found 6 existing drugs against 4 potential anti-COVID-19 targets (CCL20, CSF3, CXCL1, CXCL10) that might have novel anti-COVID-19 indications. Finally, these drug targets were computationally prioritized based on gene ranking algorithms, which revealed CXCL10 as the common and strongest candidate with 2 existing drugs. Furthermore, the list of 283 SARS-CoV-2-associated proteins could be valuable not only as anti-COVID-19 targets but also useful for COVID-19 biomarker development.

Inhalations with thermal waters in respiratory diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Inhalations with thermal waters are an old therapeutic method used in the therapy of respiratory diseases as a treatment of choice showing a long-lasting outcome with no side effects. Paradoxically, there is little well-established research on their mechanisms of action. AIM OF THE STUDY: The aim of this paper is therefore to summarize the influence of inhalatory treatment with thermal waters on the main symptoms and features of respiratory disorders including allergy-like symptoms, inflammation, oxidant-anti-oxidant balance, cellular influx, disturbed mucus secretions, recurrent infections, pulmonary and nasal function and quality of life. A short history of inhalations is also presented. MATERIALS AND METHODS: The present paper is a sum-up of research articles on the use of inhalations with thermal waters in respiratory disorders. RESULTS: According to the herein presented literature, the use of thermal water inhalations is beneficial for almost all manifestations of respiratory diseases. The mode of their action remains still unclear; however, it seems that the most important one relies on the restoration of proper defense mechanisms of the organism. CONCLUSIONS: Inhalations with thermal waters alleviate symptoms of respiratory diseases. They also improve the quality of life of the patients and seem to be a good add-on therapy in the treatment of disorders of the respiratory system.

The therapeutic potential of inorganic polyphosphate: A versatile physiological polymer to control coronavirus disease (COVID-19).

Rationale: The pandemic caused by the novel coronavirus SARS-CoV-2 is advancing rapidly. In particular, the number of severe courses of the disease is still dramatically high. An efficient drug therapy that helps to improve significantly the fatal combination of damages in the airway epithelia, in the extensive pulmonary microvascularization and finally multiorgan failure, is missing. The physiological, inorganic polymer, polyphosphate (polyP) is a molecule which could prevent the initial phase of the virus life cycle, the attachment of the virus to the target cells, and improve the epithelial integrity as well as the mucus barrier. Results: Surprisingly, polyP matches perfectly with the cationic groove on the RBD. Subsequent binding studies disclosed that polyP, with a physiological chain length of 40 phosphate residues, abolishes the binding propensity of the RBD to the ACE2 receptor. In addition to this first mode of action of polyP, this polymer causes in epithelial cells an increased gene expression of the major mucins in the airways, of MUC5AC and MUC1, as well as a subsequent glycoprotein production. MUC5AC forms a gel-like mucus layer trapping inhaled particles which are then transported out of the airways, while MUC1 constitutes the periciliary liquid layer and supports ciliary beating. As a third mode of action, polyP undergoes enzymatic hydrolysis of the anhydride bonds in the airway system by alkaline phosphatase, releasing metabolic energy. Conclusions: This review summarizes the state of the art of the biotherapeutic potential of the polymer polyP and the findings from basic research and outlines future biomedical applications.

Effect of Korean Red Ginseng and Rg3 on Asian Sand Dust-Induced MUC5AC, MUC5B, and MUC8 Expression in Bronchial Epithelial Cells.

Korean Red ginseng (KRG), commonly used in traditional medicine, has anti-inflammatory, anti- oxidative, and anti-tumorigenic properties. Asian sand dust (ASD) is known to aggravate upper and lower airway inflammatory responses. BEAS-2B cells were exposed to ASD with or without KRG or ginsenoside Rg3. Mucin 5AC (MUC5AC), MUC5B, and MUC8 mRNA and protein expression levels were determined using quantitative RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor kappa B (NF-κB), activator protein 1, and mitogen-activated protein kinase expression and activity were determined using western blot analysis. ASD induced MUC5AC, MUC5B, and MUC8 mRNA and protein expression in BEAS-2B cells, which was significantly inhibited by KRG and Rg3. Although ASD-induced mucin expression was associated with NF-κB and p38 mitogen-activated protein kinase (MAPK) activity, KRG and Rg3 significantly suppressed only ASD-induced NF-κB expression and activity. KRG and Rg3 inhibited ASD-induced mucin gene expression and protein production from bronchial epithelial cells. These results suggest that KRG and Rg3 have potential for treating mucus-producing airway inflammatory diseases.

The MAPKinase Signaling and the Stimulatory Protein-1 (Sp1) Transcription Factor Are Involved in the Phototherapy Effect on Cytokines Secretion from Human Bronchial Epithelial Cells Stimulated with Cigarette Smoke Extract.

The present study was aimed to investigate the phototherapy effect with low-level laser on human bronchial epithelial cells activated by cigarette smoke extract (CSE). Phototherapy has been reported to actuate positively for controlling the generation/release of anti-inflammatory and pro-inflammatory mediators from different cellular type activated by distinct stimuli. It is not known whether the IL-8 and IL-10 release from CSE-stimulated human bronchial epithelium (BEAS) cells can be influenced by phototherapy. Human bronchial epithelial cell (BEAS) line was cultured in a medium with CSE and irradiated (660 nm) at 9 J. Apoptosis index was standardized with Annexin V and the cellular viability was evaluated by MTT. IL-8, IL-10, cAMP, and NF-κB were measured by ELISA as well as the Sp1, JNK, ERK1/2, and p38MAPK. Phototherapy effect was studied in the presence of mithramycin or the inhibitors of JNK or ERK. The IL-8, cAMP, NF-κB, JNK, p38, and ERK1/2 were downregulated by phototherapy. Both the JNK and the ERK inhibitors potentiated the phototherapy effect on IL-8 as well as on cAMP secretion from BEAS. On the contrary, IL-10 and Sp1 were upregulated by phototherapy. The mithramycin blocked the phototherapy effect on IL-10. The results suggest that phototherapy has a dual effect on BEAS cells because it downregulates the IL-8 secretion by interfering with CSE-mediated signaling pathways, and oppositely upregulates the IL-10 secretion through of Sp1 transcription factor. The manuscript provides evidence that the phototherapy can interfere with MAPK signaling via cAMP in order to attenuate the IL-8 secretion from CSE-stimulated BEAS. In addition, the present study showed that phototherapy effect is driven to downregulation of the both the IL-8 and the ROS secretion and at the same time the upregulation of IL-10 secretion. Besides it, the increase of Sp-1 transcription factor was crucial for laser effect in upregulating the IL-10 secretion. The dexamethasone corticoid produces a significant inhibitory effect on IL-8 as well as ROS secretion, but on the other hand, the corticoid blocked the IL-10 secretion. Taking it into consideration, it is reasonable to suggest that the beneficial effect of laser therapy on lung diseases involves its action on unbalance between pro-inflammatory and anti-inflammatory mediators secreted by human bronchial epithelial cells through different signaling pathway.

Retinoic acid improves baseline barrier function and attenuates TNF-α-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o.

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward "band-shift" in occludin Western immunoblots. The presence of RA partially reversed TNF-α's effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a "cytokine storm" may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

In search of preventive strategies: novel high-CBD Cannabis sativa extracts modulate ACE2 expression in COVID-19 gateway tissues.

With the current COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for new therapies and prevention strategies that can help curtail disease spread and reduce mortality. The inhibition of viral entry and thus spread is a plausible therapeutic avenue. SARS-CoV-2 uses receptor-mediated entry into a human host via the angiotensin-converting enzyme 2 (ACE2), which is expressed in lung tissue as well as the oral and nasal mucosa, kidney, testes and gastrointestinal tract. The modulation of ACE2 levels in these gateway tissues may be an effective strategy for decreasing disease susceptibility. Cannabis sativa, especially those high in the anti-inflammatory cannabinoid cannabidiol (CBD), has been found to alter gene expression and inflammation and harbour anti-cancer and anti-inflammatory properties. However, its effects on ACE2 expression remain unknown. Working under a Health Canada research license, we developed over 800 new C. sativa cultivars and hypothesized that high-CBD C. sativa extracts may be used to down-regulate ACE2 expression in target COVID-19 tissues. Using artificial 3D human models of oral, airway and intestinal tissues, we identified 13 high-CBD C. sativa extracts that decrease ACE2 protein levels. Some C. sativa extracts down-regulate serine protease TMPRSS2, another critical protein required for SARS-CoV-2 entry into host cells. While our most effective extracts require further large-scale validation, our study is important for future analyses of the effects of medical cannabis on COVID-19. The extracts of our most successful novel high-CBD C. sativa lines, pending further investigation, may become a useful and safe addition to the prevention/treatment of COVID-19 as an adjunct therapy.

The herbal extract EPs® 7630 increases the antimicrobial airway defense through monocyte-dependent induction of IL-22 in T cells.

The phytotherapeutic compound EPs® 7630, an extract manufactured from Pelargonium sidoides roots, is frequently used for the treatment of airway infections. Nevertheless, the knowledge of the mode of action of EPs® 7630 is still sparse. Our study aimed at further elucidating the underlying pharmacological mechanisms by focusing on antimicrobial defense mechanisms of EPs® 7630. While investigating the influence of EPs® 7630 on lymphokine production by PBMCs, we found that EPs® 7630 is a novel inducer of IL-22 and IL-17. This cytokine-inducing effect was most pronounced for IL-22 and clearly dose-dependent starting from 1 µg/ml of the extract. Furthermore, EPs® 7630 pretreatment selectively enhanced the IL-22 and IL-17 production capacity of CD3/28-activated PBMCs while strongly limiting the IFN-γ production capacity of innate lymphoid cells. The relevance of EPs® 7630-induced IL-22 production was proven in vitro and in vivo, where IL-22 provoked a strong increase of the antimicrobial protein S100A9 in lung epithelial cells and pulmonary tissue, respectively. A detailed analysis of IL-22 induction modi revealed no direct influence of EPs® 7630 on the basal or anti-CD3/CD28 antibody-induced IL-22 production by CD4+ memory T cells. In fact, EPs® 7630-induced IL-22 production by CD4+ memory T cells was found to be essentially dependent on soluble mediators (IL-1/IL-23) as well as on direct cellular contact with monocytes. In summary, our study reveals a new immune-modulating function of EPs® 7630 that might confer IL-22 and IL-17-induced protection from bacterial airway infection. KEY MESSAGES: EPs® 7630 selectively strengthens IL-22 and IL-17 production of memory T cells. EPs® 7630 limits the IFN-y production capacity of innate lymphoid cells. EPs® 7630-caused IL-22 production by T cells is essentially dependent on monocytes. IL-22 increase antimicrobial proteins (AMPs) in airway epithelium. EPs® 7630 might protect against airway infection by induction of AMP-inducers.

Impact of the Local Inflammatory Environment on Mucosal Vitamin D Metabolism and Signaling in Chronic Inflammatory Lung Diseases.

Vitamin D plays an active role in the modulation of innate and adaptive immune responses as well as in the protection against respiratory pathogens. Evidence for this immunomodulatory and protective role is derived from observational studies showing an association between vitamin D deficiency, chronic airway diseases and respiratory infections, and is supported by a range of experimental studies using cell culture and animal models. Furthermore, recent intervention studies have now shown that vitamin D supplementation reduces exacerbation rates in vitamin D-deficient patients with chronic obstructive pulmonary disease (COPD) or asthma and decreases the incidence of acute respiratory tract infections. The active vitamin D metabolite, 1,25-dihydroxy-vitamin D (1,25(OH)2D), is known to contribute to the integrity of the mucosal barrier, promote killing of pathogens (via the induction of antimicrobial peptides), and to modulate inflammation and immune responses. These mechanisms may partly explain its protective role against infections and exacerbations in COPD and asthma patients. The respiratory mucosa is an important site of local 1,25(OH)2D synthesis, degradation and signaling, a process that can be affected by exposure to inflammatory mediators. As a consequence, mucosal inflammation and other disease-associated factors, as observed in e.g., COPD and asthma, may modulate the protective actions of 1,25(OH)2D. Here, we discuss the potential consequences of various disease-associated processes such as inflammation and exposure to pathogens and inhaled toxicants on vitamin D metabolism and local responses to 1,25(OH)2D in both immune- and epithelial cells. We furthermore discuss potential consequences of disturbed local levels of 25(OH)D and 1,25(OH)2D for chronic lung diseases. Additional insight into the relationship between disease-associated mechanisms and local effects of 1,25(OH)2D is expected to contribute to the design of future strategies aimed at improving local levels of 1,25(OH)2D and signaling in chronic inflammatory lung diseases.

Respiratory Syncytial Virus-Induced Oxidative Stress Leads to an Increase in Labile Zinc Pools in Lung Epithelial Cells.

Zinc supplementation in cell culture has been shown to inhibit various viruses, like herpes simplex virus, rotavirus, severe acute respiratory syndrome (SARS) coronavirus, rhinovirus, and respiratory syncytial virus (RSV). However, whether zinc plays a direct antiviral role in viral infections and whether viruses have adopted strategies to modulate zinc homeostasis have not been investigated. Results from clinical trials of zinc supplementation in infections indicate that zinc supplementation may be beneficial in a pathogen- or disease-specific manner, further underscoring the importance of understanding the interaction between zinc homeostasis and virus infections at the molecular level. We investigated the effect of RSV infection on zinc homeostasis and show that RSV infection in lung epithelial cells leads to modulation of zinc homeostasis. The intracellular labile zinc pool increases upon RSV infection in a multiplicity of infection (MOI)-dependent fashion. Small interfering RNA (siRNA)-mediated knockdown of the ubiquitous zinc uptake transporter ZIP1 suggests that labile zinc levels are increased due to the increased uptake by RSV-infected cells as an antiviral response. Adding zinc to culture medium after RSV infection led to significant inhibition of RSV titers, whereas depletion of zinc by a zinc chelator, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) led to an increase in RSV titers. The inhibitory effect of zinc was specific, as other divalent cations had no effect on RSV titers. Both RSV infection and zinc chelation by TPEN led to reactive oxygen species (ROS) induction, whereas addition of zinc blocked ROS induction. These results suggest a molecular link between RSV infection, zinc homeostasis, and oxidative-stress pathways and provide new insights for developing strategies to counter RSV infection.IMPORTANCE Zinc deficiency rates in developing countries range from 20 to 30%, and zinc supplementation trials have been shown to correct clinical manifestations attributed to zinc deficiency, but the outcomes in the case of respiratory infections have been inconsistent. We aimed at understanding the role of zinc homeostasis in respiratory syncytial virus (RSV) infection. Infection of lung epithelial cell lines or primary small-airway epithelial cells led to an increase in labile zinc pools, which was due to increased uptake of zinc. Zinc supplementation inhibited RSV replication, whereas zinc chelation had an opposing effect, leading to increases in RSV titers. Increases in labile zinc in RSV-infected cells coincided with induction of reactive oxygen species (ROS). Both zinc depletion and addition of exogenous ROS led to enhanced RSV infection, whereas addition of the antioxidant inhibited RSV, suggesting that zinc is part of an interplay between RSV-induced oxidative stress and the host response to maintain redox balance.

Sanguinarine Rapidly Relaxes Rat Airway Smooth Muscle Cells Dependent on TAS2R Signaling.

Excessive contraction of airway smooth muscle cells (ASMCs) is a hallmark feature of asthma. Intriguing, the activation of bitter taste receptor (TAS2R) in ASMCs can relax ASMCs. However, there is a lack of potent TAS2R agonists that can be used in asthma therapies since those tested agonists cannot relax ASMCs at the dose below a few hundred micromolar. Considering that sanguinarine (SA) is a bitter substance often used in small doses for the treatment of asthma in folk medicine, the present study was to determine the rapid relaxation effect of SA on ASMCs and to reveal the underlying mechanisms associated with TAS2R signaling. Here, cell stiffness, traction force, calcium signaling, cAMP levels, and the mRNA expression were evaluated by using optical magnetic twisting cytometry, traction force microscopy, Fluo-4/AM labeling, enzyme-linked immunosorbent assay (ELISA), and quantitative (q)RT-PCR, respectively. We found that 0.5 µM SA immediately decreased cell stiffness and traction force, which is comparable with the effect of 5 µM isoproterenol. In addition, 0.5 µM SA immediately increased intracellular free calcium concentration ([Ca2+]i) and decreased the mRNA expression of contractile proteins such as calponin and α-smooth muscle actin after the treatment for 24 h. Furthermore, SA-mediated decrease in cell stiffness/traction force and increase in [Ca2+]i were significantly blunted by inhibiting the TAS2Rs signaling. These findings establish the rapid relaxation effect of SA at low concentration (<1 µM) on cultured ASMCs depending on TAS2R signaling, indicating that SA might be developed as a useful bronchodilator in asthma therapy.

Ginsenoside Rg3 ameliorates acute exacerbation of COPD by suppressing neutrophil migration.

Acute Exacerbation of Chronic Obstructive Pulmonary Disease (AECOPD) is an irreversible inflammatory airways disease responsible for global health burden, involved with a complex condition of immunological change. Exacerbation-mediated neutrophilia is an important factor in the pathogenesis of cigarette smoke-induced AECOPD. Ginsenoside Rg3, a red-ginseng-derived compound, has multiple pharmacological properties such as anti-inflammatory and antitumor activities. Here, we investigated a protective role of Rg3 against AECOPD, focusing on neutrophilia. 14-week-cigarette smoke (CS) exposure and non-typeable Haemophilus inflenzae (NTHi) infection were used to establish the AECOPD murine model. Rg3 (10, 20, 40 mg/kg) was administered intragastrically from the 12th week of CS exposure before infection, and this led to improved lung function and lung morphology, and reduced neutrophilic inflammation, indicating a suppressive effect on neutrophil infiltration by Rg3. Further investigations on the mechanism of Rg3 on neutrophils were carried out using bronchial epithelial cell (BEAS-2B) and neutrophil co-culture and transepithelial migration model. Pre-treatment of neutrophils with Rg3 reduced neutrophil migration, which seemed to be the result of inhibition of phosphatidylinositol (PtdIns) 3-kinases (PI3K) activation within neutrophils. Thus, Rg3 could inhibit exacerbation-induced neutrophilia in COPD by negatively regulating PI3K activities in neutrophils. This study provides a potential natural drug against AECOPD neutrophil inflammation.

Arsenic represses airway epithelial mucin expression by affecting retinoic acid signaling pathway.

Arsenic is a ubiquitous environmental toxicant, found in high concentrations worldwide. Although abundant research has dealt with arsenic-induced cancers, studies on mechanisms of non-malignant lung diseases have not been complete. In addition, decades of research have mostly concentrated on high-dose arsenic exposure, which has very limited use in modeling the biological effects of today's low-dose exposures. Indeed, accumulated evidence has shown that low-dose arsenic exposure (i.e. ≤100 ppb) may also alter lung homeostasis by causing host susceptibility to viral infection. However, the underlying mechanism of this alteration is unknown. In this study, we found that low-dose sodium arsenite (As (III)) repressed major airway mucins-MUC5AC and MUC5B at both mRNA and protein levels. We further demonstrated that this repression was not caused by cellular toxicity or mediated by the reduction of a common mucin-inducing pathway-EGFR. Other established mucin activators- dsRNA, IL1ß or IL17 were not able to override As (III)-induced mucin repression. Interestingly, the suppressing effect of As (III) appeared to be partially reversible, and supplementation of all trans retinoic acid (t-RA) doses dependently restored mucin gene expression. Further analyses indicated that As (III) treatment significantly reduced the protein level of retinoic acid receptors (RARα, γ and RXRα) as well as RARE promoter reporter activity. Therefore, our study fills in an important knowledge gap in the field of low-dose arsenic exposure. The interference of RA signaling, and mucin gene expression may be important pathogenic factors in low-dose arsenic induced lung toxicity.

Peptide Glycodendrimers as Potential Vaccines for Olive Pollen Allergy.

Olive pollen is one of the most important causes of respiratory allergy, with Ole e 1 being the most clinically relevant sensitizing allergen. Peptide-based vaccines represent promising therapeutic approaches, but the use of adjuvants is required to strengthen the weak immunogenicity of small peptides. We propose the use of dendrimeric scaffolds conjugated to the T cell immunodominant epitope of Ole e 1 (OE109-130) for the development of novel vaccines against olive pollen allergy. Four dendrimeric scaffolds containing an ester/ether with nine mannoses, an ester succinimidyl linker with nine N-acetyl-glucosamine units or nine ethylene glycol units conjugated to OE109-130 peptide were designed, and their cytotoxicity, internalization pattern, and immunomodulatory properties were analyzed in vitro. None of the dendrimers exhibited cytotoxicity in humanized rat basophil (RBL-2H3), human bronchial epithelial Calu-3, and human mast LAD2 cell lines. Confocal images indicated that mannosylated glycodendropeptides exhibited lower colocalization with a lysosomal marker. Moreover, mannosylated glycodendropeptides showed higher transport tendency through the epithelial barrier formed by Calu-3 cells cultured at the air-liquid interface. Finally, mannosylated glycodendropeptides promoted Treg and IL10+Treg proliferation and IL-10 secretion by peripheral blood mononuclear cells from allergic patients. Mannosylated dendrimers conjugated with OE109-130 peptide from Ole e 1 have been identified as suitable candidates for the development of novel vaccines of olive pollen allergy.

Inula helenium and Grindelia squarrosa as a source of compounds with anti-inflammatory activity in human neutrophils and cultured human respiratory epithelium.

ETHNOPHARMACOLOGICAL RELEVANCE: During the epidemic season, over 90% of acute wheezing disease is associated with bronchial inflammation. Both neutrophil- and eosinophil-mediated inflammation have been involved in the pathophysiology of acute bronchitis, but neutrophil cell recruitment has been shown to be dominant. The ongoing inflammation increases the chemotaxis of neutrophils to inflamed site providing to their overaccumulation. The pharmacological reduction of neutrophil migration can be limited by suppression of major chemo-attractants and cytokines (IL-8, IL-1ß and TNF-α) release and downregulation of adhesive molecules. AIM OF THE STUDY: During a screening of plants traditionally used in respiratory tracts diseases (e.g. cough, rhinitis, bronchitis, throat infection, fever, influenza) in Europe, we have selected roots of Inula helenium and aerial parts of Grindelia squarrosa as a potential source of compounds limiting neutrophil migration. MATERIALS AND METHODS: The effect on IL-8, IL-1ß and TNF-α release by neutrophils and respiratory epithelium cell line (A549) was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesive molecules was analyzed with flow cytometry, and the neutrophil attachment to the epithelial cells was assessed fluorimetrically. RESULTS: We confirmed the ability of selected extracts and compounds to suppress neutrophil binding to the epithelium surface via downregulation of ß2 integrin. Alantolactone and grindelic acid have shown significant suppression of IL-8, TNF-α and IL-1ß release comparable with budesonide, used as a positive control. CONCLUSIONS: The present study demonstrated that Inula helenium and Grindelia squarrosa, which have been traditionally used in Europe as medicinal plants, are a valuable source of active compounds with anti-inflammatory activity. Our observations justify the traditional use of I. helenium and G. squarrosa for a treatment of inflammation-based diseases in respiratory tract.

Ergosterol attenuates cigarette smoke extract-induced COPD by modulating inflammation, oxidative stress and apoptosis in vitro and in vivo.

Cigarette smoke (CS) is the major cause of chronic obstructive pulmonary disease (COPD). CS heightens inflammation, oxidative stress and apoptosis. Ergosterol is the main bioactive ingredient in Cordyceps sinensis (C. sinensis), a traditional medicinal herb for various diseases. The objective of this work was to investigate the effects of ergosterol on anti-inflammatory and antioxidative stress as well as anti-apoptosis in a cigarette smoke extract (CSE)-induced COPD model both in vitro and in vivo Our results demonstrate that CSE induced inflammatory and oxidative stress and apoptosis with the involvement of the Bcl-2 family proteins via the nuclear factor kappa B (NF-κB)/p65 pathway in both 16HBE cells and Balb/c mice. CSE induced epithelial cell death and increased the expression of nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), malondialdehyde (MAD) and the apoptosis-related proteins cleaved caspase 3/7/9 and cleaved-poly-(ADP)-ribose polymerase (PARP) both in vitro and in vivo, whereas decreased the levels of superoxide dismutase (SOD) and catalase (CAT). Treatment of 16HBE cells and Balb/c mice with ergosterol inhibited CSE-induced inflammatory and oxidative stress and apoptosis by inhibiting the activation of NF-κB/p65. Ergosterol suppressed apoptosis by inhibiting the expression of the apoptosis-related proteins both in vitro and in vivo Moreover, the usage of QNZ (an inhibitor of NF-κB) also partly demonstrated that NF-κB/p65 pathway was involved in the ergosterol protective progress. These results show that ergosterol suppressed COPD inflammatory and oxidative stress and apoptosis through the NF-κB/p65 pathway, suggesting that ergosterol may be partially responsible for the therapeutic effects of cultured C. sinensis on COPD patients.

Molecular Signature of Asthma-Enhanced Sensitivity to CuO Nanoparticle Aerosols from 3D Cell Model.

More than 5% of any population suffers from asthma, and there are indications that these individuals are more sensitive to nanoparticle aerosols than the healthy population. We used an air-liquid interface model of inhalation exposure to investigate global transcriptomic responses in reconstituted three-dimensional airway epithelia of healthy and asthmatic subjects exposed to pristine (nCuO) and carboxylated (nCuOCOOH) copper oxide nanoparticle aerosols. A dose-dependent increase in cytotoxicity (highest in asthmatic donor cells) and pro-inflammatory signaling within 24 h confirmed the reliability and sensitivity of the system to detect acute inhalation toxicity. Gene expression changes between nanoparticle-exposed versus air-exposed cells were investigated. Hierarchical clustering based on the expression profiles of all differentially expressed genes (DEGs), cell-death-associated DEGs (567 genes), or a subset of 48 highly overlapping DEGs categorized all samples according to "exposure severity", wherein nanoparticle surface chemistry and asthma are incorporated into the dose-response axis. For example, asthmatics exposed to low and medium dose nCuO clustered with healthy donor cells exposed to medium and high dose nCuO, respectively. Of note, a set of genes with high relevance to mucociliary clearance were observed to distinctly differentiate asthmatic and healthy donor cells. These genes also responded differently to nCuO and nCuOCOOH nanoparticles. Additionally, because response to transition-metal nanoparticles was a highly enriched Gene Ontology term (FDR 8 × 10-13) from the subset of 48 highly overlapping DEGs, these genes may represent biomarkers to a potentially large variety of metal/metal oxide nanoparticles.

Antitussive, expectorant, and anti-inflammatory effects of Adenophorae Radix powder in ICR mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Adenophora triphylla var. japonica is frequently used as an oriental medicinal plant in Korea, China, and Japan for its anti-inflammatory, antitussive, and hepatoprotective effects. AIM OF THE STUDY: In the present study, the antitussive, expectorant, and anti-inflammatory effects of AR powder were investigated using animal models to evaluate their potential to treat respiratory disorders. MATERIALS AND METHODS: AR powder was administered orally to mice once daily for 11 days, at dose levels of 400, 200, and 100 mg/kg. Theobromine (TB), ambroxol (AM) and dexamethasone (DEXA) were used as standard drugs for antitussive effects, expectorant effects and anti-inflammatory effects, respectively. Evaluations of antitussive effects were based on changes in body weight, the number of cough responses and the histopathology of the lung and trachea. Expectorant effects were based on changes in the body weight, macroscopic observations of body surface redness, the mucous secretion of the trachea and histopathology of lung (secondary bronchus). Anti-inflammatory effects were based on changes in the body weight, macroscopic observations involving redness and edema of the treated ear, absolute and relative ear weights and histopathology of the treated ears. RESULTS: Allergic acute inflammation and coughing induced by exposure to NH4OH and symptoms of xylene-induced contact dermatitis were significantly inhibited by treatment with AR powder in a dose-dependent manner. Histological analyses revealed that AR powder decreased the OD values in trachea lavage fluid, reduced body surface redness, thicknesses of intrapulmonary secondary bronchus mucosa, and the number of PAS-positive mucous producing cells. Overall, AR powder administered at 200 mg/kg displayed superior antitussive and expectorant effects as compared to TB (50 mg/kg), and AM (250 mg/kg). At the highest concentration (400 mg/kg) AR powder displayed only moderately improved anti-inflammatory activities as compared to DEXA (1 mg/kg). CONCLUSION: The results obtained in this study suggest that AR powder exerts dose-dependent, favorable antitussive, expectorant, and anti-inflammatory activities achieved through modulation of the activity of mast cells and respiratory mucous production. Therefore, AR powder may serve as a therapeutic agent in various respiratory disorders, especially those that occur as a result of environmental toxicants.