Sulforaphane prevents and reverses allergic airways disease in mice via anti-inflammatory, antioxidant, and epigenetic mechanisms.

Sulforaphane has been investigated in human pathologies and preclinical models of airway diseases. To provide further mechanistic insights, we explored L-sulforaphane (LSF) in the ovalbumin (OVA)-induced chronic allergic airways murine model, with key hallmarks of asthma. Histological analysis indicated that LSF prevented or reversed OVA-induced epithelial thickening, collagen deposition, goblet cell metaplasia, and inflammation. Well-known antioxidant and anti-inflammatory mechanisms contribute to the beneficial effects of LSF. Fourier transform infrared microspectroscopy revealed altered composition of macromolecules, following OVA sensitization, which were restored by LSF. RNA sequencing in human peripheral blood mononuclear cells highlighted the anti-inflammatory signature of LSF. Findings indicated that LSF may alter gene expression via an epigenetic mechanism which involves regulation of protein acetylation status. LSF resulted in histone and α-tubulin hyperacetylation in vivo, and cellular and enzymatic assays indicated decreased expression and modest histone deacetylase (HDAC) inhibition activity, in comparison with the well-known pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Molecular modeling confirmed interaction of LSF and LSF metabolites with the catalytic domain of metal-dependent HDAC enzymes. More generally, this study confirmed known mechanisms and identified potential epigenetic pathways accounting for the protective effects and provide support for the potential clinical utility of LSF in allergic airways disease.

Analysis and comparison of anti-RBD neutralizing antibodies from AZD-1222, Sputnik V, Sinopharm and Covaxin vaccines and its relationship with gender among health care workers.

BACKGROUND: Vaccine efficiency has a significant role in the public perception of vaccination. The current study was designed to evaluate the efficacy of COVID-19 vaccines (AZD-1222, Sputnik-V, Sinopharm, and Covaxin) and the effect of gender on vaccine efficacy. We evaluated the efficacy of these vaccines among 214 health care employees in Iran. Blood samples were taken from all participants on day 0 and 14 days after the second dose. Humoral responses were evaluated by the PT-SARS-CoV-2-Neutralizing-Ab-96. RESULTS: The frequency of immunized individuals in the Sputnik V and AZD-1222 groups was 91% and 86%, respectively. This rate was 61% and 67% for Sinopharm and Covaxin vaccines. A comparison of the results obtained from the effectiveness of the vaccines between female and male groups did not demonstrate a significant difference. CONCLUSION: According to the results, Sputnik V and AZD-1222 vaccines were more effective than Sinopharm and Covaxin vaccines. Moreover, the effectiveness of these vaccines is not related to gender.

Imbalance of the renin-angiotensin system may contribute to inflammation and fibrosis in IBD: a novel therapeutic target?

OBJECTIVE: We evaluated the influence of the renin-angiotensin system (RAS) on intestinal inflammation and fibrosis. DESIGN: Cultured human colonic myofibroblast proliferation and collagen secretion were assessed following treatment with angiotensin (Ang) II and Ang (1-7), their receptor antagonists candesartan and A779, and the ACE inhibitor captopril. Circulating and intestinal RAS components were evaluated in patients with and without IBD. Disease outcomes in patients with IBD treated with ACE inhibitors and angiotensin receptor blockers (ARBs) were assessed in retrospective studies. RESULTS: Human colonic myofibroblast proliferation was reduced by Ang (1-7) in a dose-dependent manner (p<0.05). Ang II marginally but not significantly increased proliferation, an effect reversed by candesartan (p<0.001). Colonic myofibroblast collagen secretion was reduced by Ang (1-7) (p<0.05) and captopril (p<0.001), and was increased by Ang II (p<0.001). Patients with IBD had higher circulating renin (mean 25.4 vs 18.6 mIU/L, p=0.026) and ACE2:ACE ratio (mean 0.92 vs 0.69, p=0.015) than controls without IBD. RAS gene transcripts and peptides were identified in healthy and diseased bowels. Colonic mucosal Masson's trichrome staining correlated with Ang II (r=0.346, p=0.010) and inversely with ACE2 activity (r=-0.373, p=0.006). Patients with IBD who required surgery (1/37 vs 12/75, p=0.034) and hospitalisation (0/34 vs 8/68, p=0.049) over 2 years were less often treated with ACE inhibitors and ARBs than patients not requiring surgery or hospitalisation. CONCLUSIONS: The RAS mediates fibrosis in human cell cultures, is expressed in the intestine and perturbed in intestinal inflammation, and agents targeting this system are associated with improved disease outcomes.

iPSC- and mesenchymoangioblast-derived mesenchymal stem cells provide greater protection against experimental chronic allergic airways disease compared with a clinically used corticosteroid.

The airway remodeling (AWR) associated with chronic allergic airways disease (AAD)/asthma contributes to irreversible airway obstruction. This study compared and combined the antiremodeling and other effects of induced pluripotent stem cell and mesenchymoangioblast-derived mesenchymal stem cells (MCA-MSCs) with the corticosteroid dexamethasone (Dex) in experimental chronic AAD/asthma. Female BALB/c mice subjected to 11 wk of ovalbumin (Ova)-induced chronic AAD were intranasally administered MCA-MSCs (1 × 106 cells/mouse; once weekly on wk 10 and 11), Dex (0.5 mg/ml; once daily for 2 wk), or both combined. MCA-MSC detection and changes in airway inflammation (AI), AWR, and airway hyperresponsiveness (AHR) were measured at the end of wk 11. Mice with chronic AAD had significant AI, goblet cell metaplasia, epithelial damage/thickening, aberrant TGF-ß1 levels, subepithelial myofibroblast accumulation, airway/lung fibrosis, and AHR (all P < 0.001 vs. healthy controls). MCA-MSCs were detected in the lungs up to 5-7 d postadministration and demonstrated modest anti-inflammatory but striking antifibrotic effects against Ova-induced AAD, effectively decreasing AHR by 70-75% (all P < 0.05 vs. Ova alone). In comparison, Dex predominantly demonstrated anti-inflammatory effects, decreasing AHR by ∼30%. Combining MCA-MSCs with Dex provided equivalent protection to that offered by either therapy alone. MCA-MSCs reduce chronic AAD-induced AWR and AHR to a greater extent than Dex and may act as a suitable adjunct therapy to corticosteroid treatment of asthma.-Royce, S. G., Mao, W., Lim, R., Kelly, K., Samuel, C. S. iPSC- and mesenchymoangioblast-derived mesenchymal stem cells provide greater protection against experimental chronic allergic airways disease compared with a clinically used corticosteroid.

A novel Approach for Non-Invasive Lung Imaging and Targeting Lung Immune Cells.

Despite developments in pulmonary radiotherapy, radiation-induced lung toxicity remains a problem. More sensitive lung imaging able to increase the accuracy of diagnosis and radiotherapy may help reduce this problem. Super-paramagnetic iron oxide nanoparticles are used in imaging, but without further modification can cause unwanted toxicity and inflammation. Complex carbohydrate and polymer-based coatings have been used, but simpler compounds may provide additional benefits. Herein, we designed and generated super-paramagnetic iron oxide nanoparticles coated with the neutral natural dietary amino acid glycine (GSPIONs), to support non-invasive lung imaging and determined particle biodistribution, as well as understanding the impact of the interaction of these nanoparticles with lung immune cells. These GSPIONs were characterized to be crystalline, colloidally stable, with a size of 12 ± 5 nm and a hydrodynamic diameter of 84.19 ± 18 nm. Carbon, Hydrogen, Nitrogen (CHN) elemental analysis estimated approximately 20.2 × 103 glycine molecules present per nanoparticle. We demonstrated that it is possible to determine the biodistribution of the GSPIONs in the lung using three-dimensional (3D) ultra-short echo time magnetic resonance imaging. The GSPIONs were found to be taken up selectively by alveolar macrophages and neutrophils in the lung. In addition, the GSPIONs did not cause changes to airway resistance or induce inflammatory cytokines. Alveolar macrophages and neutrophils are critical regulators of pulmonary inflammatory diseases, including allergies, infections, asthma and chronic obstructive pulmonary disease (COPD). Therefore, pulmonary Magnetic Resonance (MR) imaging and preferential targeting of these lung resident cells by our nanoparticles offer precise imaging tools, which can be utilized to develop precision targeted radiotherapy as well as diagnostic tools for lung cancer, thereby having the potential to reduce the pulmonary complications of radiation.

Intranasal administration of mesenchymoangioblast-derived mesenchymal stem cells abrogates airway fibrosis and airway hyperresponsiveness associated with chronic allergic airways disease.

Structural changes known as airway remodeling (AWR) characterize chronic/severe asthma and contribute to lung dysfunction. Thus, we assessed the in vivo efficacy of induced pluripotent stem cell and mesenchymoangioblast-derived mesenchymal stem cells (MCA-MSCs) on AWR in a murine model of chronic allergic airways disease (AAD)/asthma. Female Balb/c mice were subjected to a 9-wk model of ovalbumin (Ova)-induced chronic AAD and treated intravenously or intranasally with MCA-MSCs from weeks 9 to 11. Changes in airway inflammation (AI), AWR, and airway hyperresponsiveness (AHR) were assessed. Ova-injured mice presented with AI, goblet cell metaplasia, epithelial thickening, increased airway TGF-ß1 levels, subepithelial myofibroblast and collagen accumulation, total lung collagen concentration, and AHR (all P < 0.001 vs. uninjured control group). Apart from epithelial thickness, all other parameters measured were significantly, although not totally, decreased by intravenous delivery of MCA-MSCs to Ova-injured mice. In comparison, intranasal delivery of MCA-MSCs to Ova-injured mice significantly decreased all parameters measured (all P < 0.05 vs. Ova group) and, most notably, normalized aberrant airway TGF-ß1 levels, airway/lung fibrosis, and AHR to values measured in uninjured animals. MCA-MSCs also increased collagen-degrading gelatinase levels. Hence, direct delivery of MCA-MSCs offers great therapeutic benefit for the AWR and AHR associated with chronic AAD.-Royce, S. G., Rele, S., Broughton, B. R. S., Kelly, K., Samuel, C. S. Intranasal administration of mesenchymoangioblast-derived mesenchymal stem cells abrogates airway fibrosis and airway hyperresponsiveness associated with chronic allergic airways disease.

Synchrotron-Fourier transform infrared maps of ovalbumin-induced murine chronic allergic airways disease: Correlation with conventional histology.

Asthma is a chronic respiratory disease characterised by airway inflammation, remodeling and hyperresponsiveness. The ability to replicate these asthma traits in the well-established ovalbumin induced chronic model of allergic airways disease is an important tool for asthma research and preclinical drug development. Here, spectra derived from focal plane array and Synchrotron-Fourier transform infrared maps were used to analyse biochemical changes in lung tissue from an ovalbumin-induced murine chronic allergic airways disease model. Analysis of the chemical maps resulted in distinct clusters and significant changes in the lipid and proteins regions of the spectra between the saline control and diseased lung tissue samples. Overall, the utilisation of conventional histological methodologies and Synchrotron infrared microspectroscopy has the ability to expand the characterisation of murine models of asthma.

The Coagulant Factor Xa Induces Protease-Activated Receptor-1 and Annexin A2-Dependent Airway Smooth Muscle Cytokine Production and Cell Proliferation.

During asthma exacerbation, plasma circulating coagulant factor X (FX) enters the inflamed airways and is activated (FXa). FXa may have an important role in asthma, being involved in thrombin activation and an agonist of protease-activated receptor-1 (PAR-1). Extracellular annexin A2 and integrins are also implicated in PAR-1 signaling. In this study, the potential role of PAR-1 in mediating the effects of FXa on human airway smooth muscle (ASM) cell cytokine production and proliferation was investigated. FXa (5-50 nM), but not FX, stimulated increases in ASM IL-6 production and cell number after 24- and 48-hour incubation, respectively (P < 0.05; n = 5). FXa (15 nM) also stimulated increases in the levels of mRNA for cytokines (IL-6), cell cycle-related protein (cyclin D1), and proremodeling proteins (FGF-2, PDGF-B, CTGF, SM22, and PAI-1) after 3-hour incubation (P < 0.05; n = 4). The actions of FXa were insensitive to inhibition by hirudin (1 U/ml), a selective thrombin inhibitor, but were attenuated by SCH79797 (100 nM), a PAR-1 antagonist, or Cpd 22 (1 µM), an inhibitor of integrin-linked kinase. The selective targeting of PAR-1, annexin A2, or ß1-integrin by small interfering RNA and/or by functional blocking antibodies also attenuated FXa-evoked responses. In contrast, the targeting of annexin A2 did not inhibit thrombin-stimulated ASM function. In airway biopsies of patients with asthma, FXa and annexin A2 were detected in the ASM bundle by immunohistochemistry. These findings establish FXa as a potentially important asthma mediator, stimulating ASM function through actions requiring PAR-1 and annexin A2 and involving integrin coactivation.

Airway Remodeling and Hyperreactivity in a Model of Bronchopulmonary Dysplasia and Their Modulation by IL-1 Receptor Antagonist.

Bronchopulmonary dysplasia (BPD) is a chronic disease of extreme prematurity that has serious long-term consequences including increased asthma risk. We earlier identified IL-1 receptor antagonist (IL-1Ra) as a potent inhibitor of murine BPD induced by combining perinatal inflammation (intraperitoneal LPS to pregnant dams) and exposure of pups to hyperoxia (fraction of inspired oxygen = 0.65). In this study, we determined whether airway remodeling and hyperresponsiveness similar to asthma are evident in this model, and whether IL-1Ra is protective. During 28-day exposure to air or hyperoxia, pups received vehicle or 10 mg/kg IL-1Ra by daily subcutaneous injection. Lungs were then prepared for histology and morphometry of alveoli and airways, or for real-time PCR, or inflated with agarose to prepare precision-cut lung slices to visualize ex vivo intrapulmonary airway contraction and relaxation by phase-contrast microscopy. In pups reared under normoxic conditions, IL-1Ra treatment did not affect alveolar or airway structure or airway responses. Pups reared in hyperoxia developed a severe BPD-like lung disease, with fewer, larger alveoli, increased subepithelial collagen, and increased expression of α-smooth muscle actin and cyclin D1. After hyperoxia, methacholine elicited contraction with similar potency but with an increased maximum reduction in lumen area (air, 44%; hyperoxia, 89%), whereas dilator responses to salbutamol were maintained. IL-1Ra treatment prevented hyperoxia-induced alveolar disruption and airway fibrosis but, surprisingly, not the increase in methacholine-induced airway contraction. The current study is the first to demonstrate ex vivo airway hyperreactivity caused by systemic maternal inflammation and postnatal hyperoxia, and it reveals further preclinical mechanistic insights into IL-1Ra as a treatment targeting key pathophysiological features of BPD.

Serelaxin improves the therapeutic efficacy of RXFP1-expressing human amnion epithelial cells in experimental allergic airway disease.

Current asthma therapies primarily target airway inflammation (AI) and suppress episodes of airway hyperresponsiveness (AHR) but fail to treat airway remodelling (AWR), which can develop independently of AI and contribute to irreversible airway obstruction. The present study compared the anti-remodelling and therapeutic efficacy of human bone marrow-derived mesenchymal stem cells (MSCs) to that of human amnion epithelial stem cells (AECs) in the setting of chronic allergic airways disease (AAD), in the absence or presence of an anti-fibrotic (serelaxin; RLX). Female Balb/c mice subjected to the 9-week model of ovalbumin (OVA)-induced chronic AAD, were either vehicle-treated (OVA alone) or treated with MSCs or AECs alone [intranasally (i.n.)-administered with 1×106 cells once weekly], RLX alone (i.n.-administered with 0.8 mg/ml daily) or a combination of MSCs or AECs and RLX from weeks 9-11 (n=6/group). Measures of AI, AWR and AHR were then assessed. OVA alone exacerbated AI, epithelial damage/thickness, sub-epithelial extracellular matrix (ECM) and total collagen deposition, markers of collagen turnover and AHR compared with that in saline-treated counterparts (all P<0.01 compared with saline-treated controls). RLX or AECs (but not MSCs) alone normalized epithelial thickness and partially diminished the OVA-induced fibrosis and AHR by ∼40-50% (all P<0.05 compared with OVA alone). Furthermore, the combination treatments normalized epithelial thickness, measures of fibrosis and AHR to that in normal mice, and significantly decreased AI. Although AECs alone demonstrated greater protection against the AAD-induced AI, AWR and AHR, compared with that of MSCs alone, combining RLX with MSCs or AECs reversed airway fibrosis and AHR to an even greater extent.

Ultrathin unsedated transnasal gastroscopy in monitoring eosinophilic esophagitis.

BACKGROUND: Ultrathin unsedated transnasal gastroscopy (UTEG) has a number of advantages applicable to eosinophilic esophagitis (EoE) and has not been evaluated for this condition. AIM: The aim of the study is to determine the feasibility of UTEG in patients with EoE and the acceptability of histological specimens obtained at biopsy. METHOD: All patients with a diagnosis of EoE presenting to the outpatients department of two hospitals (Box Hill Hospital and The Alfred Hospital, Melbourne Australia) were asked to participate in the study. UTEG was performed on consenting individuals. Feasibility was determined by the success of nasal intubaton, patient perception according to post procedural survey, and adequacy of esophageal biopsies was assessed. RESULTS: Ninety-six consecutive patients with EoE were offered UTEG, and 24 agreed to participate in the study. Seventy-four UTEGs were performed over a period of 26 months (September 2012 to December 2014). Nineteen patients had repeat procedures. Successful nasal intubation occurred in 97% (72 of 74 procedures), and 21 of 24 (86%) described high satisfaction with the procedure and minimal discomfort, and would choose UTEG for future procedures. Mean duration was 5 min. Adverse events of epistaxis (three cases) and vomiting of liquid contents during the procedure (two cases) were recorded, cardiorespiratory parameters remaining normal in all patients. All completed procedures produced adequate histological samples. CONCLUSION: In those who decide to undergo UTEG, it is a safe and well-tolerated procedure.

Alteration of Airway Reactivity and Reduction of Ryanodine Receptor Expression by Cigarette Smoke in Mice.

Small airways are a major site of airflow limitation in chronic obstructive pulmonary disease (COPD). Despite the detrimental effects of long-term smoking in COPD, the effects of acute cigarette smoke (CS) exposure on small airway reactivity have not been fully elucidated. Balb/C mice were exposed to room air (sham) or CS for 4 days to cause airway inflammation. Changes in small airway lumen area in response to contractile agents were measured in lung slices in situ using phase-contrast microscopy. Separate slices were pharmacologically maintained at constant intracellular Ca(2+) using caffeine/ryanodine before contractile measurements. Gene and protein analysis of contractile signaling pathways were performed on separate lungs. Monophasic contraction to serotonin became biphasic after CS exposure, whereas contraction to methacholine was unaltered. This altered pattern of contraction was normalized by caffeine/ryanodine. Expression of contractile agonist-specific receptors was unaltered; however, all isoforms of the ryanodine receptor were down-regulated. This is the first study to show that acute CS exposure selectively alters small airway contraction to serotonin and down-regulates ryanodine receptors involved in maintaining Ca(2+) oscillations in airway smooth muscle. Understanding the contribution of ryanodine receptors to altered airway reactivity may inform the development of novel treatment strategies for COPD.

Rosiglitazone elicits in vitro relaxation in airways and precision cut lung slices from a mouse model of chronic allergic airways disease.

Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor-γ (PPARγ) ligand, is a novel dilator of small airways in mouse precision cut lung slices (PCLS). In this study, relaxation to RGZ and ß-adrenoceptor agonists were compared in trachea from naïve mice and guinea pigs and trachea and PCLS from a mouse model of chronic allergic airways disease (AAD). Airways were precontracted with methacholine before addition of PPARγ ligands [RGZ, ciglitazone (CGZ), or 15-deoxy-(Δ12,14)-prostaglandin J2 (15-deoxy-PGJ2)] or ß-adrenoceptor agonists (isoprenaline and salbutamol). The effects of T0070907 and GW9662 (PPARγ antagonists) or epithelial removal on relaxation were assessed. Changes in force of trachea and lumen area in PCLS were measured using preparations from saline-challenged mice and mice sensitized (days 0 and 14) and challenged with ovalbumin (3 times/wk, 6 wk). RGZ and CGZ elicited complete relaxation with greater efficacy than ß-adrenoceptor agonists in mouse airways but not guinea pig trachea, while 15-deoxy-PGJ2 did not mediate bronchodilation. Relaxation to RGZ was not prevented by T0070907 or GW9662 or by epithelial removal. RGZ-induced relaxation was preserved in the trachea and increased in PCLS after ovalbumin-challenge. Although RGZ was less potent than ß-adrenoceptor agonists, its effects were additive with salbutamol and isoprenaline and only RGZ maintained potency and full efficacy in maximally contracted airways or after allergen challenge. Acute PPARγ-independent, epithelial-independent airway relaxation to RGZ is resistant to functional antagonism and maintained in both trachea and PCLS from a model of chronic AAD. These novel efficacious actions of RGZ support its therapeutic potential in asthma when responsiveness to ß-adrenoceptor agonists is limited.

Mechanistic insights into the contribution of epithelial damage to airway remodeling. Novel therapeutic targets for asthma.

It has been suggested that an inherent airway epithelial repair defect is the root cause of airway remodeling in asthma. However, the relationship between airway epithelial injury and repair, airway remodeling, and airway hyperresponsiveness (AHR) has not been directly examined. We investigated the contribution of epithelial damage and repair to the development of airway remodeling and AHR using a validated naphthalene (NA)-induced murine model of airway injury. In addition, we examined the endogenous versus exogenous role of the epithelial repair peptide trefoil factor 2 (TFF2) in disease pathogenesis. A single dose of NA (200 mg/kg in 10 ml/kg body weight corn oil [CO] vehicle, intraperitoneally) was administered to mice. Control mice were treated with CO (10 ml/kg body weight, intraperitoneally). At 12, 24, 48, and 72 hours after NA or CO injection, AHR and various measures of airway remodeling were examined by invasive plethysmography and morphometric analyses, respectively. TFF2-deficient mice and intranasal treatment were used to examine the role of the epithelial repair peptide. NA treatment induced denudation and apoptosis of airway epithelial cells, goblet cell metaplasia, elevated AHR, and increased levels of endogenous TFF2. Airway epithelial changes peaked at 12 hours after NA treatment, whereas airway remodeling changes were observed from 48 hours. TFF2 was protective against epithelial damage and induced remodeling and was found to mediate organ protection via a platelet-derived growth factor-associated mechanism. Our findings directly demonstrate the contribution of epithelial damage to airway remodeling and AHR and suggest that preventing airway epithelial damage and promoting epithelial repair may have therapeutic implications for asthma treatment.

Characterization of a novel model incorporating airway epithelial damage and related fibrosis to the pathogenesis of asthma.

Asthma develops from injury to the airways/lungs, stemming from airway inflammation (AI) and airway remodeling (AWR), both contributing to airway hyperresponsiveness (AHR). Airway epithelial damage has been identified as a new etiology of asthma but is not targeted by current treatments. Furthermore, it is poorly studied in currently used animal models of AI and AWR. Therefore, this study aimed to incorporate epithelial damage/repair with the well-established ovalbumin (OVA)-induced model of chronic allergic airway disease (AAD), which presents with AI, AWR, and AHR, mimicking several features of human asthma. A 3-day naphthalene (NA)-induced model of epithelial damage/repair was superimposed onto the 9-week OVA-induced model of chronic AAD, before 6 weeks of OVA nebulization (NA+OVA group), during the second last OVA nebulization period (OVA/NA group) or 1 day after the 6-week OVA nebulization period (OVA+NA group), using 6-8-week-old female Balb/c mice (n=6-12/group). Mice subjected to the 9-week OVA model, 3-day NA model or respective vehicle treatments (saline and corn oil) were used as appropriate controls. OVA alone significantly increased epithelial thickness and apoptosis, goblet cell metaplasia, TGF-ß1, subepithelial collagen (assessed by morphometric analyses of various histological stains), total lung collagen (hydroxyproline analysis), and AHR (invasive plethysmography) compared with that in saline-treated mice (all P<0.05 vs saline treatment). NA alone caused a significant increase in epithelial denudation and apoptosis, TGF-ß1, subepithelial, and total lung collagen compared with respective measurements from corn oil-treated controls (all P<0.01 vs corn oil treatment). All three combined models underwent varying degrees of epithelial damage and AWR, with the OVA+NA model demonstrating the greatest increase in subepithelial/total lung collagen and AHR (all P<0.05 vs OVA alone or NA alone). These combined models of airway epithelial damage/AAD demonstrated that epithelial damage is a key contributor to AWR, fibrosis and related AHR, and augments the effects of AI on these parameters.

Cell cycle effects of L-sulforaphane, a major antioxidant from cruciferous vegetables: The role of the anaphase promoting complex.

L-sulforaphane (LSF) is a natural isothiocyanate found in cruciferous vegetables particularly broccoli. LSF has been identified as a potent antioxidant and anti-cancer agent and is widely known to regulate phase II detoxifying enzymes and induce cell cycle arrest or apoptosis in malignant cells in vitro and in vivo. Previous studies have found significant G2/M cell cycle arrest in response to LSF in various model of cancer and results have mainly been attributed to increased cyclin B1 protein levels and increased p21expression. Using genome-wide mRNA-Seq analysis we provide insights into the molecular mechanisms of action of LSF to identify a key pathway in cell cycle progression - the role of the anaphase promoting complex (APC) pathway. We evaluated gene expression changes in human erythroleukemic K562 cells following treatment with 15 µM LSF for 48h and compared them to immortalized human keratinocytes, human microvascular endothelial cells (HMEC-1) cells and normal human umbilical endothelial cells (HUVEC). We identified disparate gene expression changes in response to LSF between malignant and normal cells and immortalized cell lines. The results highlight significant down-regulation of kinase CDK1 which is suggestive that the existence and activity of APC/CDC20 complex will be inhibited along with its associated down-stream degradation of key cell cycle regulators preventing cell cycle progression from mitotic exit.

Trefoil factor-2 reverses airway remodeling changes in allergic airways disease.

Trefoil factor 2 (TFF2) is a small peptide with an important role in mucosal repair. TFF2 is up-regulated in asthma, suggesting a role in asthma pathogenesis. Given its known biological role in promoting epithelial repair, TFF2 might be expected to exert a protective function in limiting the progression of airway remodeling in asthma. The contribution of TFF2 to airway remodeling in asthma was investigated by examining the expression of TFF2 in the airway and lung, and evaluating the effects of recombinant TFF2 treatment on established airway remodeling in a murine model of chronic allergic airways disease (AAD). BALB/c mice were sensitized and challenged with ovalbumin (OVA) or saline for 9 weeks, whereas mice with established OVA-induced AAD were treated with TFF2 or vehicle control (intranasally for 14 d). Effects on airway remodeling, airway inflammation, and airway hyperresponsiveness were then assessed, whereas TFF2 expression was determined by immunohistochemistry. TFF2 expression was significantly increased in the airways of mice with AAD, compared with expression levels in control mice. TFF2 treatment resulted in reduced epithelial thickening, subepithelial collagen deposition, goblet-cell metaplasia, bronchial epithelium apoptosis, and airway hyperresponsiveness (all P < 0.05, versus vehicle control), but TFF2 treatment did not influence airway inflammation. The increased expression of endogenous TFF2 in response to chronic allergic inflammation is insufficient to prevent the progression of airway inflammation and remodeling in a murine model of chronic AAD. However, exogenous TFF2 treatment is effective in reversing aspects of established airway remodeling. TFF2 has potential as a novel treatment for airway remodeling in asthma.

Airway hyperresponsiveness is associated with airway remodeling but not inflammation in aging Cav1-/- mice.

BACKGROUND: Airway inflammation and airway remodeling are the key contributors to airway hyperresponsiveness (AHR), a characteristic feature of asthma. Both processes are regulated by Transforming Growth Factor (TGF)-ß. Caveolin 1 (Cav1) is a membrane bound protein that binds to a variety of receptor and signaling proteins, including the TGF-ß receptors. We hypothesized that caveolin-1 deficiency promotes structural alterations of the airways that develop with age will predispose to an increased response to allergen challenge. METHODS: AHR was measured in Cav1-deficient and wild-type (WT) mice 1 to 12 months of age to examine the role of Cav1 in AHR and the relative contribution of inflammation and airway remodeling. AHR was then measured in Cav1-/- and WT mice after an ovalbumin-allergen challenge performed at either 2 months of age, when remodeling in Cav1-/- and WT mice was equivalent, and at 6 months of age, when the Cav1-/- mice had established airway remodeling. RESULTS: Cav1-/- mice developed increased thickness of the subepithelial layer and a correspondingly increased AHR as they aged. In addition, allergen-challenged Cav1-/- mice had an increase in AHR greater than WT mice that was largely independent of inflammation. Cav1-/- mice challenged at 6 months of age have decreased AHR compared to those challenged at 2 months with correspondingly decreased BAL IL-4 and IL-5 levels, inflammatory cell counts and percentage of eosinophils. In addition, in response to OVA challenge, the number of goblet cells and α-SMA positive cells in the airways were reduced with age in response to OVA challenge in contrast to an increased collagen deposition further enhanced in absence of Cav1. CONCLUSION: A lack of Cav1 contributed to the thickness of the subepithelial layer in mice as they aged resulting in an increase in AHR independent of inflammation, demonstrating the important contribution of airway structural changes to AHR. In addition, age in the Cav1-/- mice is a contributing factor to airway remodeling in the response to allergen challenge.

Combination therapy with relaxin and methylprednisolone augments the effects of either treatment alone in inhibiting subepithelial fibrosis in an experimental model of allergic airways disease.

Although CSs (corticosteroids) demonstrate potent effects in the control of airway inflammation in asthma, many patients continue to experience symptoms and AHR (airway hyper-responsiveness) despite optimal treatment with these agents, probably due to progressive airway remodelling. Identifying novel therapies that can target airway remodelling and/or airway reactivity may improve symptom control in these patients. We have demonstrated previously that the anti-fibrotic hormone RLN (relaxin) can reverse airway remodelling (epithelial thickening and subepithelial fibrosis) and AHR in a murine model of AAD (allergic airways disease). In the present study, we compared the effects of RLN with a CS (methylprednisolone) on airway remodelling and AHR when administered independently or in combination in the mouse AAD model. Female mice at 6-8 weeks of age were sensitized and challenged to OVA (ovalbumin) over a 9-week period and treated with methylprednisolone, RLN, a combination of both treatments or vehicle controls. Methylprednisolone was administered intraperitoneally on the same day as nebulization for 6 weeks, whereas recombinant human RLN-2 was administered via subcutaneously implanted osmotic mini-pumps from weeks 9-11. RLN or methylprednisolone alone were both able to significantly decrease subepithelial thickness and total lung collagen deposition; whereas RLN but not methylprednisolone significantly decreased epithelial thickness and AHR. Additionally, combination therapy with CS and RLN more effectively reduced subepithelial collagen thickness than either therapy alone. These findings demonstrate that RLN can modulate a broader range of airway remodelling changes and AHR than methylprednisolone and the combination of both treatments offers enhanced control of subepithelial fibrosis.

Comparative Study of Ectopic Lymphoid Aggregates in Sheep and Murine Models of Bleomycin-Induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by excessive deposition of extracellular matrix in the interstitial lung parenchyma, often manifested by dyspnea and progressive loss of lung function. The role of inflammation in the pathogenesis of IPF is not well understood. This study evaluated the histopathological and inflammatory components of bleomycin-induced pulmonary fibrosis in mouse and sheep models, in terms of their ability to translate to the human IPF. Merino sheep (n = 8) were bronchoscopically administered with two bleomycin infusions, two weeks apart, into a caudal lung segment, with a saline (control) administered into a caudal segment in the opposite lung. Balb/c mice were twice intranasally instilled, one week apart, with either bleomycin (n = 7); or saline (control, n = 7). Lung samples were taken for the histopathological assessment 28 days in sheep and 21 days in mice after the first bleomycin administration. We observed tertiary lymphoid aggregates, in the fibrotic lung parenchyma of sheep, but not in mouse lung tissues exposed to bleomycin. B-cell and T-cell infiltration significantly increased in sheep lung tissues compared to mouse lung tissues due to bleomycin injury. Statistical analysis showed that the fibrotic score, fibrotic fraction, and tissue fraction significantly increased in sheep lung tissues compared to murine lung tissues. The presence of tertiary lymphoid aggregates in the lung parenchyma and increased infiltration of T-cells and B-cells, in the sheep model, may be useful for the future study of the underlying inflammatory disease mechanisms in the lung parenchyma of IPF patients.