In vivo models of lung neutrophil activation. Comparison of mice and hamsters

BACKGROUND: Evidence suggests that both the migration and activation of neutrophils into the airway is of importance in pathological conditions such as pulmonary emphysema. In the present study, we describe in vivo models of lung neutrophil infiltration and activation in mice and hamsters. RESULTS: BALB/c and C57BL/6 mice were intranasally treated with lipopolysaccharide (0.3 mg/kg). Twenty-four hours after, animals were treated intranasally with N-Formyl-Met-Leu-Phe (0 to 5 mg/kg). Golden Syrian hamsters were treated intratracheally with 0.5 mg/kg of lipopolysaccharide. Twenty-four hours after, animals were treated intratracheally with 0.25 mg/kg of N-Formyl-Met-Leu-Phe. Both mice and hamster were sacrificed two hours after the N-Formyl-Met-Leu-Phe application. In both BALB/c and C57BL/6 mice, a neutrophil infiltration was observed after the sequential application of lipopolysaccharide and N-Formyl-Met-Leu-Phe. However, 5 times less neutrophil was found in C57BL/6 mice when compared to BALB/c mice. This was reflected in the neutrophil activation parameters measured (myeloperoxidase and elastase activities). Despite the presence of neutrophil and their activation status, no lung haemorrhage could be detected in both strains of mice. When compared with mice, the lung inflammation induced by the sequential application of lipopolysaccharide and N-Formyl-Met-Leu-Phe was much greater in the hamster. In parallel with this lung inflammation, a significant lung haemorrhage was also observed. CONCLUSIONS: Both mouse and hamster can be used for pharmacological studies of new drugs or other therapeutics agents that aimed to interfere with neutrophil activation. However, only the hamster model seems to be suitable for studying the haemorrhagic lung injury proces

Long receptor residence time of C26 contributes to super agonist activity at the human β2 adrenoceptor

Super agonists produce greater functional responses than endogenous agonists in the same assay, and their unique pharmacology is the subject of increasing interest and debate. We propose that receptor residence time and the duration of receptor signaling contribute to the pharmacology of super agonism. We have further characterized the novel β2 adrenoceptor agonist C26 (7-[(R)-2-((1R,2R)-2-benzyloxycyclopentylamino)-1-hydroxyethyl]-4-hydroxybenzothiazolone), which displays higher intrinsic activity than the endogenous ligand adrenaline in cAMP accumulation, β-arrestin-2 recruitment, and receptor internalization assays. C26 recruited β-arrestin-2, and internalized the Green Fluorescent Protein (GFP)-taggedβ2 adrenoceptor at a slow rate, with half-life (t1/2) values of 0.78 ± 0.1 and 0.78 ± 0.04 hours, respectively. This was compared with 0.31 ± 0.04 and 0.34 ± 0.01 hours for adrenaline-mediated β-arrestin-2 recruitment and GFP-β2 internalization, respectively. The slower rate for C26 resulted in levels of β-arrestin-2 recruitment increasing up to 4-hour agonist incubation, at which point the intrinsic activity was determined to be 124.3 ± 0.77% of the adrenaline response. In addition to slow functional kinetics, C26 displayed high affinity with extremely slow receptor dissociation kinetics, giving a receptor residence half-life of 32.7 minutes at 37°C, which represents the slowest dissociation rate we have observed for any β2 adrenoceptor agonist tested to date. In conclusion, we propose that the gradual accumulation of long-lived active receptor complexes contributes to the increased intrinsic activity of C26 over time. This highlights the need to consider the temporal aspects of agonist binding and signaling when characterizing ligands as super agonists

Peroxisome Proliferator-Activated Receptor alpha (PPAR alpha) down-regulation in cystic fibrosis lymphocytes

Background: PPARs exhibit anti-inflammatory capacities and are potential modulators of the inflammatory response. We hypothesized that their expression and/or function may be altered in cystic fibrosis (CF), a disorder characterized by an excessive host inflammatory response. Methods: PPARα, β and γ mRNA levels were measured in peripheral blood cells of CF patients and healthy subjects via RT-PCR. PPARα protein expression and subcellular localization was determined via western blot and immunofluorescence, respectively. The activity of PPARα was analyzed by gel shift assay. Results: In lymphocytes, the expression of PPARα mRNA, but not of PPARβ, was reduced (-37%; p < 0.002) in CF patients compared with healthy persons and was therefore further analyzed. A similar reduction of PPARα was observed at protein level (-26%; p < 0.05). The transcription factor was mainly expressed in the cytosol of lymphocytes, with low expression in the nucleus. Moreover, DNA binding activity of the transcription factor was 36% less in lymphocytes of patients (p < 0.01). For PPARα and PPARβ mRNA expression in monocytes and neutrophils, no significant differences were observed between CF patients and healthy persons. In all cells, PPARγ mRNA levels were below the detection limit. Conclusion: Lymphocytes are important regulators of the inflammatory response by releasing cytokines and antibodies. The diminished lymphocytic expression and activity of PPARα may therefore contribute to the inflammatory processes that are observed in CF

Detecting G protein-coupled receptor complexes in postmortem human brain with proximity ligation assay and a Bayesian classifier

Despite the controversy regarding the existence and physiological relevance of class A G protein-coupled receptor dimerization, there is substantial evidence for functional interactions between the dopamine D2 receptor (D2R) and the adenosine A2A receptor (A2AR). A2AR-D2R complexes have been detected in rodent brains by proximity ligation assay; however, their existence in the human brain has not been demonstrated. In this study, we used Brightfield proximity ligation assay, combined with a systematic sampling and a parameter-free naive Bayesian classifier, and demonstrated proximity between the D2R and the A2AR in the adult human ventral striatum, consistent with their colocalization within complexes and the possible existence of D2R-A2AR heteromers. These methods are applicable to the relative quantification of proximity of two proteins, as well as the expression levels of individual proteins

Quantification of collagen and proteoglycan deposition in a murine model of airway remodelling

BACKGROUND: Sub-epithelial extracellular matrix deposition is a feature of asthmatic airway remodelling associated with severity of disease, decline in lung function and airway hyperresponsiveness. The composition of, and mechanisms leading to, this increase in subepithelial matrix, and its importance in the pathogenesis of asthma are unclear. This is partly due to limitations of the current models and techniques to assess airway remodelling. METHODS: In this study we used a modified murine model of ovalbumin sensitisation and challenge to reproduce features of airway remodelling, including a sustained increase in sub-epithelial matrix deposition. In addition, we have established techniques to accurately and specifically measure changes in sub-epithelial matrix deposition, using histochemical and immunohistochemical staining in conjunction with digital image analysis, and applied these to the measurement of collagen and proteoglycans. RESULTS: 24 hours after final ovalbumin challenge, changes similar to those associated with acute asthma were observed, including inflammatory cell infiltration, epithelial cell shedding and goblet cell hyperplasia. Effects were restricted to the bronchial and peribronchial regions with parenchymal lung of ovalbumin sensitised and challenged mice appearing histologically normal. By 12 days, the acute inflammatory changes had largely resolved and increased sub-epithelial staining for collagen and proteoglycans was observed. Quantitative digital image analysis confirmed the increased deposition of sub-epithelial collagen (33%, p < 0.01) and proteoglycans (32%, p < 0.05), including decorin (66%, p < 0.01). In addition, the increase in sub-epithelial collagen deposition was maintained for at least 28 days (48%, p < 0.001). CONCLUSION: This animal model reproduces many of the features of airway remodelling found in asthma and allows accurate and reproducible measurement of sub-epithelial extra-cellular matrix deposition. As far as we are aware, this is the first demonstration of increased sub-epithelial proteoglycan deposition in an animal model of airway remodelling. This model will be useful for measurement of other matrix components, as well as for assessment of the molecular mechanisms contributing to, and agents to modulate airway remodelling

Evaluation of the PPAR-γ agonist pioglitazone in mild asthma: a double-blind randomized controlled trial

Background Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a nuclear receptor that modulates inflammation in models of asthma. To determine whether pioglitazone improves measures of asthma control and airway inflammation, we performed a single-center randomized, double-blind, placebo-controlled, parallel-group trial. Methods Sixty-eight participants with mild asthma were randomized to 12 weeks pioglitazone (30 mg for 4 weeks, then 45 mg for 8 weeks) or placebo. The primary outcome was the adjusted mean forced expiratory volume in one second (FEV1) at 12 weeks. The secondary outcomes were mean peak expiratory flow (PEF), scores on the Juniper Asthma Control Questionnaire (ACQ) and Asthma Quality of Life Questionnaire (AQLQ), fractional exhaled nitric oxide (FeNO), bronchial hyperresponsiveness (PD20), induced sputum counts, and sputum supernatant interferon gamma-inducible protein-10 (IP-10), vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), and eosinophil cationic protein (ECP) levels. Study recruitment was closed early after considering the European Medicines Agency’s reports of a potential increased risk of bladder cancer with pioglitazone treatment. Fifty-five cases were included in the full analysis (FA) and 52 in the per-protocol (PP) analysis. Results There was no difference in the adjusted FEV1 at 12 weeks (-0.014 L, 95% confidence interval [CI] -0.15 to 0.12, p = 0.84) or in any of the secondary outcomes in the FA. The PP analysis replicated the FA, with the exception of a lower evening PEF in the pioglitazone group (-21 L/min, 95% CI -39 to -4, p = 0.02). Conclusions We found no evidence that treatment with 12 weeks of pioglitazone improved asthma control or airway inflammation in mild asthma

Pioglitazone is as effective as dexamethasone in a cockroach allergen-induced murine model of asthma

<p>Abstract</p> <p>Background</p> <p>While glucocorticoids are currently the most effective therapy for asthma, associated side effects limit enthusiasm for their use. Peroxisome proliferator-activated receptor-γ (PPAR-γ) activators include the synthetic thiazolidinediones (TZDs) which exhibit anti-inflammatory effects that suggest usefulness in diseases such as asthma. How the ability of TZDs to modulate the asthmatic response compares to that of glucocorticoids remains unclear, however, because these two nuclear receptor agonists have never been studied concurrently. Additionally, effects of PPAR-γ agonists have never been examined in a model involving an allergen commonly associated with human asthma.</p> <p>Methods</p> <p>We compared the effectiveness of the PPAR-γ agonist pioglitazone (PIO) to the established effectiveness of a glucocorticoid receptor agonist, dexamethasone (DEX), in a murine model of asthma induced by cockroach allergen (CRA). After sensitization to CRA and airway localization by intranasal instillation of the allergen, Balb/c mice were challenged twice at 48-h intervals with intratracheal CRA. Either PIO (25 mg/kg/d), DEX (1 mg/kg/d), or vehicle was administered throughout the period of airway CRA exposure.</p> <p>Results</p> <p>PIO and DEX demonstrated similar abilities to reduce airway hyperresponsiveness, pulmonary recruitment of inflammatory cells, serum IgE, and lung levels of IL-4, IL-5, TNF-α, TGF-β, RANTES, eotaxin, MIP3-α, Gob-5, and Muc5-ac. Likewise, intratracheal administration of an adenovirus containing a constitutively active PPAR-γ expression construct blocked CRA induction of Gob-5 and Muc5-ac.</p> <p>Conclusion</p> <p>Given the potent effectiveness shown by PIO, we conclude that PPAR-γ agonists deserve investigation as potential therapies for human asthma.</p

PPARα downregulates airway inflammation induced by lipopolysaccharide in the mouse

BACKGROUND: Inflammation is a hallmark of acute lung injury and chronic airway diseases. In chronic airway diseases, it is associated with profound tissue remodeling. Peroxisome proliferator-activated receptor-α (PPARα) is a ligand-activated transcription factor, that belongs to the nuclear receptor family. Agonists for PPARα have been recently shown to reduce lipopolysaccharide (LPS)- and cytokine-induced secretion of matrix metalloproteinase-9 (MMP-9) in human monocytes and rat mesangial cells, suggesting that PPARα may play a beneficial role in inflammation and tissue remodeling. METHODS: We have investigated the role of PPARα in a mouse model of LPS-induced airway inflammation characterized by neutrophil and macrophage infiltration, by production of the chemoattractants, tumor necrosis factor-α (TNF-α), keratinocyte derived-chemokine (KC), macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1), and by increased MMP-2 and MMP-9 activity in bronchoalveolar lavage fluid (BALF). The role of PPARα in this model was studied using both PPARα-deficient mice and mice treated with the PPARα activator, fenofibrate. RESULTS: Upon intranasal exposure to LPS, PPARα(-/- )mice exhibited greater neutrophil and macrophage number in BALF, as well as increased levels of TNF-α, KC, MIP-2 and MCP-1, when compared to PPARα(+/+ )mice. PPARα(-/- )mice also displayed enhanced MMP-9 activity. Conversely, fenofibrate (0.15 to 15 mg/day) dose-dependently reduced the increase in neutrophil and macrophage number induced by LPS in wild-type mice. In animals treated with 15 mg/day fenofibrate, this effect was associated with a reduction in TNF-α, KC, MIP-2 and MCP-1 levels, as well as in MMP-2 and MMP-9 activity. PPARα(-/- )mice treated with 15 mg/day fenofibrate failed to exhibit decreased airway inflammatory cell infiltrate, demonstrating that PPARα mediates the anti-inflammatory effect of fenofibrate. CONCLUSION: Using both genetic and pharmacological approaches, our data clearly show that PPARα downregulates cell infiltration, chemoattractant production and enhanced MMP activity triggered by LPS in mouse lung. This suggests that PPARα activation may have a beneficial effect in acute or chronic inflammatory airway disorders involving neutrophils and macrophages

Gene expression profile comparison between bone metastatic and non-metastatic prostate cancer cell lines

Astronauts suffer from cardiovascular deconditioning during space flight where they are exposed to microgravity. Alterations under real and simulated microgravity have been found e.g. in the cytoskeleton and apoptosis in endothelial cells (ECs) and smooth muscle cells (SMCs)1, 2. P2 receptors play an important role in a variety of vascular functions of ECs and SMCs. However, the functional role of purinergic signalling in ECs and SMCs under microgravity is still unclear. In this study primary ECs and SMCs were isolated from bovine aorta and characterized using specific markers. Additionally, EC growth medium collected during culture under normal gravity was used as conditioned medium for SMCs and vice versa to mimic a co-culture model. Here we show for the first time that the P2-receptor expression pattern is altered in ECs and SMCs under simulated microgravity achieved by a clinostat. Interestingly, conditioned medium compensated the alterations in the expression of specific P2-receptors. P2X7 was down-regulated in ECs after 24h clinorotation but recovered to the gene and protein expression level found under normal gravity when cultured in conditioned medium from SMCs. Our results showed an altered P2-receptor expression pattern under simulated microgravity. The paracrine effect between ECs and SMCs seems to be an important regulator of cell behaviour under altered gravity conditions. Several artificial P2-receptor ligands are already utilized as drugs. Thus it might be reasonable to consider them for drug development for astronaut treatment of cardiovascular deconditioning in the future

Downregulation of peroxisome proliferator-activated receptors (PPARs) in nasal polyposis

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR) α, βδ and γ are nuclear receptors activated by fatty acid metabolites. An anti-inflammatory role for these receptors in airway inflammation has been suggested. METHODS: Nasal biopsies were obtained from 10 healthy volunteers and 10 patients with symptomatic allergic rhinitis. Nasal polyps were obtained from 22 patients, before and after 4 weeks of local steroid treatment (fluticasone). Real-time RT-PCR was used for mRNA quantification and immunohistochemistry for protein localization and quantification. RESULTS: mRNA expression of PPARα, PPARβδ, PPARγ was found in all specimens. No differences in the expression of PPARs were obtained in nasal biopsies from patients with allergic rhinitis and healthy volunteers. Nasal polyps exhibited lower levels of PPARα and PPARγ than normal nasal mucosa and these levels were, for PPARγ, further reduced following steroid treatment. PPARγ immunoreactivity was detected in the epithelium, but also found in smooth muscle of blood vessels, glandular acini and inflammatory cells. Quantitative evaluation of the epithelial immunostaining revealed no differences between nasal biopsies from patients with allergic rhinitis and healthy volunteers. In polyps, the PPARγ immunoreactivity was lower than in nasal mucosa and further decreased after steroid treatment. CONCLUSION: The down-regulation of PPARγ, in nasal polyposis but not in turbinates during symptomatic seasonal rhinitis, suggests that PPARγ might be of importance in long standing inflammations