CHF6001 Inhibits NF-κB activation and neutrophilic recruitment in LPS-induced lung inflammation in mice

Inhibitors of phosphodiesterase 4 (PDE4) are potent anti-inflammatory agents, inhibiting the production of inflammatory mediators through the elevation of intracellular cAMP concentrations. We studied the activity of a novel PDE4 inhibitor, CHF6001, both in vitro in human cells and in vivo, using bioluminescence imaging (BLI) in mice lung inflammation. Mice transiently transfected with the luciferase gene under the control of an NF-\u3baB responsive element (NF-\u3baB-luc) have been used to assess the in vivo anti-inflammatory activity of CHF6001 in lipopolysaccharide (LPS)-induced lung inflammation. BLI as well as inflammatory cells and the concentrations of pro-inflammatory cytokines were monitored in bronchoalveolar lavage fluids (BALF) while testing in vitro its ability to affect the production of leukotriene B4 (LTB4), measured by LC/MS/MS, by LPS/LPS/N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated human blood. CHF6001 inhibited the production of LTB4 in LPS/fMLP-activated human blood at sub-nanomolar concentrations. LPS-induced an increase of BLI signal in NF-\u3baB-luc mice, and CHF6001 administered by dry powder inhalation decreased in parallel luciferase signal, cell airway infiltration, and pro-inflammatory cytokine concentrations in BALF. The results obtained provide in vitro and in vivo evidence of the anti-inflammatory activity of the potent PDE4 inhibitor CHF6001, showing that with a topical administration that closely mimics inhalation in humans, it efficiently disrupts the NF-\u3baB activation associated with LPS challenge, an effect that may be relevant for the prevention of exacerbation episodes in chronic obstructive pulmonary disease subjects

A respirable HPV-L2 dry-powder vaccine with GLA as amphiphilic lubricant and immune-adjuvant

Vaccines not requiring cold-chain storage/distribution and suitable for needle-free delivery are urgently needed. Pulmonary administration is one of the most promising non-parenteral routes for vaccine delivery. Through a multi-component excipient and spray-drying approach, we engineered highly respirable dry-powder vaccine particles containing a three-fold repeated peptide epitope derived from human papillomavirus (HPV16) minor capsid protein L2 displayed on Pyrococcus furious thioredoxin as antigen. A key feature of our engineering approach was the use of the amphiphilic endotoxin derivative glucopyranosyl lipid A (GLA) as both a coating agent enhancing particle de-aggregation and respirability as well as a built-in immune-adjuvant. Following an extensive characterization of the in vitro aerodynamic performance, lung deposition was verified in vivo by intratracheal administration in mice of a vaccine powder containing a fluorescently labeled derivative of the antigen. This was followed by a short-term immunization study that highlighted the ability of the GLA-adjuvanted vaccine powder to induce an anti-L2 systemic immune response comparable to (or even better than) that of the subcutaneously administered liquid-form vaccine. Despite the very short-term immunization conditions employed for this preliminary vaccination experiment, the intratracheally administered dry-powder, but not the subcutaneously injected liquid-state, vaccine induced consistent HPV neutralizing responses. Overall, the present data provide proof-of-concept validation of a new formulation design to produce a dry-powder vaccine that may be easily transferred to other antigens

Proteomic Fingerprint of Lung Fibrosis Progression and Response to Therapy in Bleomycin-Induced Mouse Model

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by the aberrant accumulation of extracellular matrix in the lungs. nintedanib is one of the two FDA-approved drugs for IPF treatment; however, the exact pathophysiological mechanisms of fibrosis progression and response to therapy are still poorly understood. In this work, the molecular fingerprint of fibrosis progression and response to nintedanib treatment have been investigated by mass spectrometry-based bottom-up proteomics in paraffin-embedded lung tissues from bleomycin-induced (BLM) pulmonary fibrosis mice. Our proteomics results unveiled that (i) samples clustered depending on the tissue fibrotic grade (mild, moderate, and severe) and not on the time course after BLM treatment; (ii) the dysregulation of different pathways involved in fibrosis progression such as the complement coagulation cascades, advanced glycation end products (AGEs) and their receptors (RAGEs) signaling, the extracellular matrix-receptor interaction, the regulation of actin cytoskeleton, and ribosomes; (iii) Coronin 1A (Coro1a) as the protein with the highest correlation when evaluating the progression of fibrosis, with an increased expression from mild to severe fibrosis; and (iv) a total of 10 differentially expressed proteins (padj-value ≤ 0.05 and Fold change ≤-1.5 or ≥1.5), whose abundance varied in the base of the severity of fibrosis (mild and moderate), were modulated by the antifibrotic treatment with nintedanib, reverting their trend. Notably, nintedanib significantly restored lactate dehydrogenase B (Ldhb) expression but not lactate dehydrogenase A (Ldha). Notwithstanding the need for further investigations to validate the roles of both Coro1a and Ldhb, our findings provide an extensive proteomic characterization with a strong relationship with histomorphometric measurements. These results unveil some biological processes in pulmonary fibrosis and drug-mediated fibrosis therapy

Monitoring inflammation and airway remodeling by fluorescence molecular tomography in a chronic asthma model

Background: Asthma is a multifactorial disease for which a variety of mouse models have been developed. A major drawback of these models is represented by the transient nature of the airway pathology peaking 24-72h after challenge and resolving in 1-2weeks. We characterized the temporal evolution of pulmonary inflammation and tissue remodeling in a recently described mouse model of chronic asthma (8week treatment with 3 allergens: Dust mite, Ragweed, and Aspergillus; DRA). Methods: We studied the DRA model taking advantage of fluorescence molecular tomography (FMT) imaging using near-infrared probes to non-invasively evaluate lung inflammation and airway remodeling. At 4, 6, 8 or 11weeks, cathepsin- and metalloproteinase-dependent fluorescence was evaluated in vivo. A subgroup of animals, after 4weeks of DRA, was treated with Budesonide (100\u3bcg/kg intranasally) daily for 4weeks. Results: Cathepsin-dependent fluorescence in DRA-sensitized mice resulted significantly increased at 6 and 8weeks, and was markedly inhibited by budesonide. This fluorescent signal well correlated with ex vivo analysis such as bronchoalveolar lavage eosinophils and pulmonary inflammatory cell infiltration. Metalloproteinase-dependent fluorescence was significantly increased at 8 and 11weeks, nicely correlated with collagen deposition, as evaluated histologically by Masson's Trichrome staining, and airway epithelium hypertrophy, and was only partly inhibited by budesonide. Conclusions: FMT proved suitable for longitudinal studies to evaluate asthma progression, showing that cathepsin activity could be used to monitor inflammatory cell infiltration while metalloproteinase activity parallels airway remodeling, allowing the determination of steroid treatment efficacy in a chronic asthma model in mice

Azithromycin inhibits nuclear factor-κB activation during lung inflammation : an in vivo imaging study

We studied in vivo the potential involvement of nuclear factor-\u3baB (NF-\u3baB) pathway in the molecular mechanism of the anti-inflammatory and immunomodulatory activity of azithromycin in the lung. Mice transiently transfected with the luciferase gene under the control of a NF-\u3baB responsive element were used to assess in vivo NF-\u3baB activation by bioluminescence imaging. Bioluminescence as well as inflammatory cells and concentrations of proinflammatory cytokines in bronchoalveolar lavage fluids, were monitored in an acute model of pulmonary inflammation resulting from intratracheal instillation of lipopolysaccharide. Lipopolysaccharide (LPS) instillation induced a marked increase in lung bioluminescence in mice transiently transfected with the luciferase gene under the control of an NF-\u3baB responsive element, with significant luciferase expression in resident cells such as endothelial and epithelial cells, as assessed by duoplex immunofluorescence staining. Activation of NF-\u3baB and inflammatory cell lung infiltration linearly correlated when different doses of bortezomib were used to inhibit NF-\u3baB activation. Pretreatment with azithromycin significantly decreased lung bioluminescence and airways cell infiltration induced by LPS, also reducing proinflammatory cytokines concentrations in bronchoalveolar lavages and inhibiting NF-\u3baB nuclear translocation. The results obtained using a novel approach to monitor NF-\u3baB activation, provided, for the first time, in vivo evidence that azithromycin treatment results in pulmonary anti-inflammatory activity associated with the inhibition of NF-\u3baB activation in the lung

In Vivo Imaging of Transiently Transgenized Mice with a Bovine Interleukin 8 (CXCL8) Promoter/Luciferase Reporter Construct

One of the most remarkable properties of interleukin 8 (CXCL8/IL-8), a chemokine with known additional functions also in angiogenesis and tissue remodeling, is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by several folds in response to proinflammatory cytokines, bacterial or viral products, and cellular stress. Although mouse cells do not bear a clear homologous IL-8 gene, the murine transcriptional apparatus may well be capable of activating or repressing a heterologous IL-8 gene promoter driving a reporter gene. In order to induce a transient transgenic expression, mice were systemically injected with a bovine IL-8 promoter–luciferase construct. Subsequently mice were monitored for luciferase expression in the lung by in vivo bioluminescent image analysis over an extended period of time (up to 60 days). We demonstrate that the bovine IL-8 promoter–luciferase construct is transiently and robustly activated 3–5 hours after LPS and TNF-α instillation into the lung, peaking at 35 days after construct delivery. Bovine IL-8 promoter–luciferase activation correlates with white blood cell and neutrophil infiltration into the lung. This study demonstrates that a small experimental rodent model can be utilized for non-invasively monitoring, through a reporter gene system, the activation of an IL-8 promoter region derived from a larger size animal (bovine). This proof of principle study has the potential to be utilized also for studying primate IL-8 promoter regions

Functional Analysis of the Two Brassica AP3 Genes Involved in Apetalous and Stamen Carpelloid Phenotypes

The Arabidopsis homeotic genes APETALA3 (AP3) and PISTILLATA (PI) are B genes which encode MADS-box transcription factors and specify petal and stamen identities. In the current study, the stamen carpelloid (SC) mutants, HGMS and AMS, of B. rapa and B. napus were investigated and two types of AP3 genes, B.AP3.a and B.AP3.b, were functional characterized. B.AP3.a and B.AP3.b share high similarity in amino acid sequences except for 8 residues difference located at the C-terminus. Loss of this 8 residues in B.AP3.b led to the change of PI-derived motifs. Meanwhile, B.AP3.a specified petal and stamen development, whereas B.AP3.b only specified stamen development. In B. rapa, the mutations of both genes generated the SC mutant HGMS. In B. napus that contained two B.AP3.a and two B.AP3.b, loss of the two B.AP3.a functions was the key reason for the apetalous mutation, however, the loss-of-function in all four AP3 was related to the SC mutant AMS. We inferred that the 8 residues or the PI-derived motif in AP3 gene probably relates to petal formation

Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging

For in vivo multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH2-NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in deep tissue. Using a spectral unmixing algorithm tool it is possible to distinguish each spectral contribution. Ultimately, this enzyme pair can expand the near-infrared bioluminescent toolbox to enable rapid visualization of multiple biological processes in deep tissue using a single substrate.Optical Imaging; Biological Services; Biophysic

The Space of Stability Conditions on Abelian Threefolds, and on some Calabi-Yau Threefolds

We describe a connected component of the space of stability conditions on abelian threefolds, and on Calabi-Yau threefolds obtained as (the crepant resolution of) a finite quotient of an abelian threefold. Our proof includes the following essential steps: 1. We simultaneously strengthen a conjecture by the first two authors and Toda, and prove that it follows from a more natural and seemingly weaker statement. This conjecture is a Bogomolov-Gieseker type inequality involving the third Chern character of "tilt-stable" two-term complexes on smooth projective threefolds; we extend it from complexes of tilt-slope zero to arbitrary tilt-slope. 2. We show that this stronger conjecture implies the so-called support property of Bridgeland stability conditions, and the existence of an explicit open subset of the space of stability conditions. 3. We prove our conjecture for abelian threefolds, thereby giving reproving and generalizing a result by Maciocia and Piyaratne. Important in our approach is a more systematic understanding on the behaviour of quadratic inequalities for semistable objects under wall-crossing, closely related to the support property.Comment: 45 pages, 1 figure. v2: addressed referee comments. To appear in Inventiones Mat