Targeting Neutrophilic Inflammation Using Polymersome-Mediated Cellular Delivery.

Neutrophils are key effector cells in inflammation and play an important role in neutralizing invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before they are removed by macrophages, but if apoptosis is delayed, neutrophils can cause extensive tissue damage and chronic disease. Promotion of neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, yet neutrophils have proven difficult cells to manipulate experimentally. In this study, we deliver therapeutic compounds to neutrophils using biocompatible, nanometer-sized synthetic vesicles, or polymersomes, which are internalized by binding to scavenger receptors and subsequently escape the early endosome through a pH-triggered disassembly mechanism. This allows polymersomes to deliver molecules into the cell cytosol of neutrophils without causing cellular activation. After optimizing polymersome size, we show that polymersomes can deliver the cyclin-dependent kinase inhibitor (R)-roscovitine into human neutrophils to promote apoptosis in vitro. Finally, using a transgenic zebrafish model, we show that encapsulated (R)-roscovitine can speed up inflammation resolution in vivo more efficiently than the free drug. These results show that polymersomes are effective intracellular carriers for drug delivery into neutrophils. This has important consequences for the study of neutrophil biology and the development of neutrophil-targeted therapeutics.

Mid-infrared multispectral tissue imaging using a chalcogenide fiber supercontinuum source.

We present, to the best of our knowledge, the first demonstration of mid-infrared supercontinuum (SC) tissue imaging at wavelengths beyond 5 µm using a fiber-coupled SC source spanning 2-7.5 µm. The SC was generated in a tapered large-mode-area chalcogenide photonic crystal fiber in order to obtain broad bandwidth, high average power, and single-mode output for diffraction-limited imaging performance. Tissue imaging was demonstrated in transmission at selected wavelengths between 5.7 (1754 cm-1) and 7.3 µm (1370 cm-1) by point scanning over a sub-millimeter region of colon tissue, and the results were compared to images obtained from a commercial instrument.

A novel lung explant model for the ex vivo study of efficacy and mechanisms of anti-influenza drugs.

Influenza A virus causes considerable morbidity and mortality largely because of a lack of effective antiviral drugs. Viral neuraminidase inhibitors, which inhibit viral release from the infected cell, are currently the only approved drugs for influenza, but have recently been shown to be less effective than previously thought. Growing resistance to therapies that target viral proteins has led to increased urgency in the search for novel anti-influenza compounds. However, discovery and development of new drugs have been restricted because of differences in susceptibility to influenza between animal models and humans and a lack of translation between cell culture and in vivo measures of efficacy. To circumvent these limitations, we developed an experimental approach based on ex vivo infection of human bronchial tissue explants and optimized a method of flow cytometric analysis to directly quantify infection rates in bronchial epithelial tissues. This allowed testing of the effectiveness of TVB024, a vATPase inhibitor that inhibits viral replication rather than virus release, and to compare efficacy with the current frontline neuraminidase inhibitor, oseltamivir. The study showed that the vATPase inhibitor completely abrogated epithelial cell infection, virus shedding, and the associated induction of proinflammatory mediators, whereas oseltamivir was only partially effective at reducing these mediators and ineffective against innate responses. We propose, therefore, that this explant model could be used to predict the efficacy of novel anti-influenza compounds targeting diverse stages of the viral replication cycle, thereby complementing animal models and facilitating progression of new drugs into clinical trials.

Multidimensional endotyping in patients with severe asthma reveals inflammatory heterogeneity in matrix metalloproteinases and chitinase 3-like protein 1.

BACKGROUND: Disease heterogeneity in patients with severe asthma and its relationship to inflammatory mechanisms remain poorly understood. OBJECTIVE: We aimed to identify and replicate clinicopathologic endotypes based on analysis of blood and sputum parameters in asthmatic patients. METHODS: One hundred ninety-four asthmatic patients and 21 control subjects recruited from 2 separate centers underwent detailed clinical assessment, sputum induction, and phlebotomy. One hundred three clinical, physiologic, and inflammatory parameters were analyzed by using topological data analysis and Bayesian network analysis. RESULTS: Severe asthma was associated with anxiety and depression, obesity, sinonasal symptoms, decreased quality of life, and inflammatory changes, including increased sputum chitinase 3-like protein 1 (YKL-40) and matrix metalloproteinase (MMP) 1, 3, 8, and 12 levels. Topological data analysis identified 6 clinicopathobiologic clusters replicated in both geographic cohorts: young, mild paucigranulocytic; older, sinonasal disease; obese, high MMP levels; steroid resistant TH2 mediated, eosinophilic; mixed granulocytic with severe obstruction; and neutrophilic, low periostin levels, severe obstruction. Sputum IL-5 levels were increased in patients with severe particularly eosinophilic forms, whereas IL-13 was suppressed and IL-17 levels did not differ between clusters. Bayesian network analysis separated clinical features from intricately connected inflammatory pathways. YKL-40 levels strongly correlated with neutrophilic asthma and levels of myeloperoxidase, IL-8, IL-6, and IL-6 soluble receptor. MMP1, MMP3, MMP8, and MMP12 levels were associated with severe asthma and were correlated positively with sputum IL-5 levels but negatively with IL-13 levels. CONCLUSION: In 2 distinct cohorts we have identified and replicated 6 clinicopathobiologic clusters based on blood and induced sputum measures. Our data underline a disconnect between clinical features and underlying inflammation, suggest IL-5 production is relatively steroid insensitive, and highlight the expression of YKL-40 in patients with neutrophilic inflammation and the expression of MMPs in patients with severe asthma.

Multidimensional endotypes of asthma: topological data analysis of cross-sectional clinical, pathological, and immunological data.

BACKGROUND: Incomplete understanding of mechanisms and clinicopathobiological heterogeneity in asthma hinders research progress. Pathogenic roles for T-helper-type 17 (Th17) cells and invariant T cells implied by murine data have yet to be assessed in man. We aimed to investigate the role of Th17 and mucosal associated invariant T (MAIT) cells in airway inflammation; to characterise associations between diverse clinical and immunological features of asthma; and to identify novel multidimensional asthma endotypes. METHODS: In this single-centre, cross-sectional observational study in the UK, we assessed volunteers with mild-to-severe asthma and healthy non-atopic controls using clinical and physiological assessment and immunological sampling of blood, induced sputum, endobronchial biopsy, and bronchoalveolar lavage for flow cytometry and multiplex-electrochemiluminescence assays. Primary outcomes were changes in frequencies of Th17 and MAIT cells between health and asthma using Mann-Whitney U tests and the Jonckheere-Terpstra test (linear trend across ranked groups). The study had 80% power to detect 60% differences in T-cell frequencies at p<0·05. Bayesian Network Analysis (BNA) was used to explore associations between parameters. Topological Data Analysis (TDA) was used to identify multidimensional endotypes. The study had local research ethics approval. All participants provided informed consent. FINDINGS: Participants were 84 male and female volunteers (60 with mild-to-severe asthma and 24 healthy, non-atopic controls) aged 18-70 years recruited from clinics and research cohorts. Th17 cells and γδ17 cells were not associated with asthma, even in severe neutrophilic forms. MAIT-cell frequencies were strikingly reduced in asthma compared with health (median frequency in blood 0·9% of CD3+ cells [IQR 0·3-1·8] in asthma vs 1·6 [1·2-2·6] in health, p=0·005; in sputum 1·1 [0·7-2·0] vs 1·8 [1·6-2·3], p=0·002; and in biopsy samples 1·3 [0·7-2·3] vs 3·9% [1·3-5·3%], p=0·02), especially in severe asthma where BAL regulatory T cells were also reduced compared with those in health (4·4, 3·1-6·1, vs 8·1, 5·6-10; p=0·02). BNA and TDA identified six novel clinicopathobiological clusters of underlying disease mechanisms, with elevated mast cell mediators tryptase (p<0·0001), chymase (p=0·02), and carboxypeptidase A3 (p=0·02) in severe asthma. INTERPRETATION: This study suggests that Th17 cells do not have a major pathogenic role in human asthma. We describe a novel deficiency of MAIT cells in severe asthma. We also provide proof of concept for application of TDA to identification of multidimensional clinicopathobiological endotypes. Endotypes will require validation in further cohorts. FUNDING: Wellcome Trust.

Innate and adaptive T cells in asthmatic patients: Relationship to severity and disease mechanisms.

BACKGROUND: Asthma is a chronic inflammatory disease involving diverse cells and mediators whose interconnectivity and relationships to asthma severity are unclear. OBJECTIVE: We performed a comprehensive assessment of TH17 cells, regulatory T cells, mucosal-associated invariant T (MAIT) cells, other T-cell subsets, and granulocyte mediators in asthmatic patients. METHODS: Sixty patients with mild-to-severe asthma and 24 control subjects underwent detailed clinical assessment and provided induced sputum, endobronchial biopsy, bronchoalveolar lavage, and blood samples. Adaptive and invariant T-cell subsets, cytokines, mast cells, and basophil mediators were analyzed. RESULTS: Significant heterogeneity of T-cell phenotypes was observed, with levels of IL-13-secreting T cells and type 2 cytokines increased at some, but not all, asthma severities. TH17 cells and γδ-17 cells, proposed drivers of neutrophilic inflammation, were not strongly associated with asthma, even in severe neutrophilic forms. MAIT cell frequencies were strikingly reduced in both blood and lung tissue in relation to corticosteroid therapy and vitamin D levels, especially in patients with severe asthma in whom bronchoalveolar lavage regulatory T-cell numbers were also reduced. Bayesian network analysis identified complex relationships between pathobiologic and clinical parameters. Topological data analysis identified 6 novel clusters that are associated with diverse underlying disease mechanisms, with increased mast cell mediator levels in patients with severe asthma both in its atopic (type 2 cytokine-high) and nonatopic forms. CONCLUSION: The evidence for a role for TH17 cells in patients with severe asthma is limited. Severe asthma is associated with a striking deficiency of MAIT cells and high mast cell mediator levels. This study provides proof of concept for disease mechanistic networks in asthmatic patients with clusters that could inform the development of new therapies.

Sigmatropic Rearrangement of Vinyl Aziridines: Expedient Synthesis of Cyclic Sulfoximines from Chiral Sulfinimines.

A novel rearrangement of 2-vinyl aziridine 2-carboxylates to unusual chiral cyclic sulfoximines is described herein. The method allows the synthesis of substituted cyclic sulfoximines in high yields with complete stereocontrol, and tolerates a wide substrate scope. A one-pot process starting directly from sulfinimines provides access to complex chiral sulfoximines in only two steps from commercially available aldehydes. A mechanistic hypothesis and synthetic application in the formal synthesis of trachelanthamidine, by transformation of a cyclic sulfoximine into a pyrroline, is also disclosed.

Characterization of TRIF selectivity in the AGP class of lipid A mimetics: role of secondary lipid chains.

TLR4 agonists that favor TRIF-dependent signaling and the induction of type 1 interferons may have potential as vaccine adjuvants with reduced toxicity. CRX-547 (4), a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of lipid A mimetics possessing three (R)-3-decanoyloxytetradecanoyl groups and d-relative configuration in the aglycon, selectively reduces MyD88-dependent signaling resulting in TRIF-selective signaling, whereas the corresponding secondary ether lipid 6a containing (R)-3-decyloxytetradecanoyl groups does not. In order to determine which secondary acyl groups are important for the reduction in MyD88-dependent signaling activity of 4, the six possible ester/ether hybrid derivatives of 4 and 6a were synthesized and evaluated for their ability to induce NF-κB in a HEK293 cell reporter assay. An (R)-3-decanoyloxytetradecanoyl group on the 3-position of the d-glucosamine unit was found to be indispensable for maintaining low NF-κB activity irrespective of the substitutions (decyl or decanoyl) on the other two secondary positions. These results suggest that the carbonyl group of the 3-secondary lipid chain may impede homodimerization and/or conformational changes in the TLR4-MD2 complex necessary for MyD88 binding and pro-inflammatory cytokine induction.

NOMAD spectrometer on the ExoMars trace gas orbiter mission: part 1--design, manufacturing and testing of the infrared channels.

NOMAD is a spectrometer suite on board ESA's ExoMars trace gas orbiter due for launch in January 2016. NOMAD consists of two infrared channels and one ultraviolet and visible channel allowing the instrument to perform observations quasi-constantly, by taking nadir measurements at dayside and nightside, and during solar occultations. In this paper, the design, manufacturing, and testing of the two infrared channels are described. We focus upon the optical working principle in these channels, where an echelle grating, used as a diffractive element, is combined with an acousto-optical tunable filter, used as a diffraction order sorter.

Mid-infrared supercontinuum generation to 12.5µm in large NA chalcogenide step-index fibres pumped at 4.5µm.

We present numerical modeling of mid-infrared (MIR) supercontinuum generation (SCG) in dispersion-optimized chalcogenide (CHALC) step-index fibres (SIFs) with exceptionally high numerical aperture (NA) around one, pumped with mode-locked praseodymium-doped (Pr(3+)) chalcogenide fibre lasers. The 4.5um laser is assumed to have a repetition rate of 4MHz with 50ps long pulses having a peak power of 4.7kW. A thorough fibre design optimisation was conducted using measured material dispersion (As-Se/Ge-As-Se) and measured fibre loss obtained in fabricated fibre of the same materials. The loss was below 2.5dB/m in the 3.3-9.4µm region. Fibres with 8 and 10µm core diameters generated an SC out to 12.5 and 10.7µm in less than 2m of fibre when pumped with 0.75 and 1kW, respectively. Larger core fibres with 20µm core diameters for potential higher power handling generated an SC out to 10.6µm for the highest NA considered but required pumping at 4.7kW as well as up to 3m of fibre to compensate for the lower nonlinearities. The amount of power converted into the 8-10µm band was 7.5 and 8.8mW for the 8 and 10µm fibres, respectively. For the 20µm core fibres up to 46mW was converted.

Phenotypic characterization of lung macrophages in asthmatic patients: overexpression of CCL17.

BACKGROUND: Studies with monocyte-derived macrophages (MDMs) and animal models have suggested a role for alternatively activated (M2) macrophages in asthmatic inflammation, but in vivo evidence for this phenotype in human asthma is lacking. OBJECTIVE: To characterize the phenotype of lung macrophages from asthmatic patients in relation to disease severity and treatment. METHODS: M2 biomarkers were first identified by using MDMs exposed to T(H)2 cytokines and then used to phenotype sputum and bronchoalveolar lavage (BAL) macrophages from 12 healthy control subjects, 12 patients with mild asthma, and 14 patients with moderate asthma and to assess the effects of corticosteroids and phosphatidylinositol 3-kinase (PI3K) inhibitors. RESULTS: Sputum macrophages from asthmatic patients expressed significantly more CCL17 mRNA but less CD163 than macrophages from healthy subjects. However, none of the other M2 biomarkers were differentially expressed in asthmatic patients, and ex vivo BAL cells spontaneously produced similar amounts of M2 cytokines/chemokines (IL-10, CCL17, and CCL22). CCL17 mRNA overexpression correlated weakly but significantly with sputum eosinophilia (P = .0252) and was also observed in macrophages from patients with moderate asthma treated with inhaled steroids, suggesting relative insensitivity to inhibition by corticosteroids. The PI3K inhibitor LY294002 inhibited basal CCL17 release from BAL cells and IL-4-stimulated release from MDMs. CONCLUSIONS: This study does not support the existence in human asthma of the full M2 phenotype described to date but points to upregulation of CCL17 in both patients with mild and those with moderate asthma, providing a further source for this ligand of CCR4(+) cells that contributes to airways inflammation. CCL17 expression is corticosteroid resistant but suppressed by PI3K enzyme inhibitors.

Efficacy of Fractionated Erbium Laser in Treating Radiation-Induced Dermal Atrophy: A Case Report.

Radiation dermatitis is a common side effect of radiotherapy in patients with acute and chronic changes affecting the skin. While acute changes occur within 90 days of radiation exposure, chronic changes manifest thereafter. This paper presents a case of a 70-year-old male with squamous cell carcinoma in situ (SCCIS) on the right zygoma who was treated with superficial radiation therapy (SRT), which resulted in a hypo-pigmented atrophic scar. The scar was successfully treated with a single session of Erbium:YAG laser therapy. The findings highlight the need for improved treatment options for radiation-induced skin changes and demonstrate the efficacy of fractionated laser therapy in addressing SRT-induced dermal atrophy.

5-HT2AR and NMDAR psychedelics induce similar hyper-synchronous states in the rat cognitive-limbic cortex-basal ganglia system.

The profound changes in perception and cognition induced by psychedelic drugs are thought to act on several levels, including increased glutamatergic activity, altered functional connectivity and an aberrant increase in high-frequency oscillations. To bridge these different levels of observation, we have here performed large-scale multi-structure recordings in freely behaving rats treated with 5-HT2AR psychedelics (LSD, DOI) and NMDAR psychedelics (ketamine, PCP). While interneurons and principal cells showed disparate firing rate modulations for the two classes of psychedelics, the local field potentials revealed a shared pattern of synchronized high-frequency oscillations in the ventral striatum and several cortical areas. Remarkably, the phase differences between structures were close to zero, corresponding to <1 ms delays. Likely, this hypersynchrony has major effects on the integration of information across neuronal systems and we propose that it is a key contributor to changes in perception and cognition during psychedelic drug use. Potentially, similar mechanisms could induce hallucinations and delusions in psychotic disorders and would constitute promising targets for new antipsychotic treatments.

The RNA binding proteins ZFP36L1 and ZFP36L2 are dysregulated in airway epithelium in human and a murine model of asthma.

Introduction: Asthma is the most common chronic inflammatory disease of the airways. The airway epithelium is a key driver of the disease, and numerous studies have established genome-wide differences in mRNA expression between health and asthma. However, the underlying molecular mechanisms for such differences remain poorly understood. The human TTP family is comprised of ZFP36, ZFP36L1 and ZFP36L2, and has essential roles in immune regulation by determining the stability and translation of myriad mRNAs encoding for inflammatory mediators. We investigated the expression and possible role of the tristetraprolin (TTP) family of RNA binding proteins (RBPs), poorly understood in asthma. Methods: We analysed the levels of ZFP36, ZFP36L1 and ZFP36L2 mRNA in several publicly available asthma datasets, including single cell RNA-sequencing. We also interrogated the expression of known targets of these RBPs in asthma. We assessed the lung mRNA expression and cellular localization of Zfp36l1 and Zfp36l2 in precision cut lung slices in murine asthma models. Finally, we determined the expression in airway epithelium of ZFP36L1 and ZFP36L2 in human bronchial biopsies and performed rescue experiments in primary bronchial epithelium from patients with severe asthma. Results: We found ZFP36L1 and ZFP36L2 mRNA levels significantly downregulated in the airway epithelium of patients with very severe asthma in different cohorts (5 healthy vs. 8 severe asthma; 36 moderate asthma vs. 37 severe asthma on inhaled steroids vs. 26 severe asthma on oral corticoids). Integrating several datasets allowed us to infer that mRNAs potentially targeted by these RBPs are increased in severe asthma. Zfp36l1 was downregulated in the lung of a mouse model of asthma, and immunostaining of ex vivo lung slices with a dual antibody demonstrated that Zfp36l1/l2 nuclear localization was increased in the airway epithelium of an acute asthma mouse model, which was further enhanced in a chronic model. Immunostaining of human bronchial biopsies showed that airway epithelial cell staining of ZFP36L1 was decreased in severe asthma as compared with mild, while ZFP36L2 was upregulated. Restoring the levels of ZFP36L1 and ZFP36L2 in primary bronchial epithelial cells from patients with severe asthma decreased the mRNA expression of IL6, IL8 and CSF2. Discussion: We propose that the dysregulation of ZFP36L1/L2 levels as well as their subcellular mislocalization contributes to changes in mRNA expression and cytoplasmic fate in asthma.

Temporal interleukin-1beta secretion from primary human peripheral blood monocytes by P2X7-independent and P2X7-dependent mechanisms.

The processing and regulated secretion of IL-1beta are critical points of control of the biological activity of this important pro-inflammatory cytokine. IL-1beta is produced by both monocytes and macrophages, but the rate and mechanism of release differ according to the differentiation status and the origin of these cells. We aimed to study the control of processing and release in human blood monocytes and human monocyte-derived macrophages. Toll-like receptor (TLR)-induced IL-1beta production and release were investigated for dependence upon caspase-1, P2X7 receptor activation, and loss of membrane asymmetry associated with microvesicle shedding. TLR agonists induced P2X7 receptor-dependent IL-1beta release in both monocytes and macrophages; however, only monocytes also showed P2X7 receptor-independent release of mature IL-1beta. Furthermore, in monocytes ATP-mediated PS exposure could be activated independently of IL-1beta production. Release of IL-1beta from monocytes showed selectivity for specific TLR agonists and was accelerated by P2X7 receptor activation. Human monocytes released more IL-1beta/cell than macrophages. These data have important implications for inflammatory diseases that involve monocyte activation and IL-1 release.

Identification of lipocalin and apolipoprotein A1 as biomarkers of chronic obstructive pulmonary disease.

RATIONALE: Much effort is being made to discover noninvasive biomarkers of chronic airway disease that might enable better management, predict prognosis, and provide new therapeutic targets. OBJECTIVES: To undertake a comprehensive, unbiased proteomic analysis of induced sputum and identify novel noninvasive biomarkers for chronic obstructive pulmonary disease (COPD). METHODS: Induced sputum was obtained from patients with COPD with a spectrum of disease severity and from control subjects. Two-dimensional gel electrophoresis and mass spectrometric identification of differentially expressed proteins were first applied to induced sputum from patients with GOLD stage 2 COPD and healthy smoker control subjects. Initial results thus obtained were validated by a combination of immunoassays (Western blotting and ELISA) applied to a large subject cohort. The biomarkers were localized to bronchial mucosa by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: Of 1,325 individual protein spots identified, 37 were quantitatively and 3 qualitatively different between the two groups (P < 0.05%). Forty protein spots were subjected to tandem mass spectrometry, which identified 15 separate protein species. Seven of these were further quantified in induced sputum from 97 individuals. Using this sequential approach, two of these potential biomarkers (apolipoprotein A1 and lipocalin-1) were found to be significantly reduced in patients with COPD when compared with healthy smokers. Their levels correlated with FEV(1)/FVC, indicating their relationship to disease severity. CONCLUSIONS: A potential role for apolipoprotein A1 and lipocalin-1 in innate defense has been postulated previously; our discovery of their reduction in COPD indicates a deficient innate defense system in airway disease that could explain increased susceptibility to infectious exacerbations.

Usability and Robustness of the Wixela Inhub Dry Powder Inhaler.

Background: Wixela Inhub is a generic version of Advair Diskus recently approved by the U.S. Food and Drug Administration. The Inhub inhaler delivers fluticasone propionate (FP)/salmeterol in a dry powder formulation. The goals of our studies were to demonstrate that the Inhub inhaler can be used by representative end users and confirm the robustness of the Inhub inhaler. Methods: Study 1: A nondosing usability assessment, the device orientation study, confirmed that intended users (represented by patients diagnosed with asthma or chronic obstructive pulmonary disease [COPD] who were naive to dry powder inhalers and current Advair Diskus users) could use the Inhub inhaler safely and effectively. Subjects were provided with an Inhub inhaler in commercial packaging, including instructions for use, and were asked to undertake three dose simulations using the inhaler. Subjects were encouraged to interact with this new drug delivery device as they would at home. Subjects were not provided with training on the use of the device. Subjects were observed interacting with the Inhub inhaler, and those who currently use Diskus were also observed interacting with the Diskus to determine whether their mental model of the use of Diskus impacted their interaction with the Inhub device, this assessment was not a primary outcome of the study. Study 2: This is an open-label clinical study to confirm the robustness of the Inhub inhaler after at home patient use. Subjects diagnosed with asthma or COPD were provided Inhub inhaler training and subsequently self-administered 3 weeks of twice daily doses of Wixela Inhub 250 µg FP/50 µg salmeterol in the home environment. The Inhub inhalers were returned to the investigator after ∼3 weeks of outpatient use for in vitro tests on the drug remaining in each inhaler. Results: Study 1 enrolled 110 subjects, and all completed the study. Most subjects (100/110) held the Inhub inhaler in the correct orientation and of those who did not, 9 still achieved a peak inhalation flow rate of ≥30 L/min and a total inhaled volume of ≥1 L, thus meeting the requirements of the study success criteria. In Study 2, 111 pediatric, adult, and elderly subjects with asthma or COPD received the study drug. After ∼3 weeks of outpatient use of the Inhub inhaler by subjects, comprehensive in vitro testing demonstrated that the FP and salmeterol pharmaceutical performance in the Inhub inhaler was preserved. Conclusions: The majority of subjects demonstrated safe and effective use of the Inhub inhaler. In vitro testing and inspections confirmed the robustness of the Inhub inhaler after outpatient use. Clinical trial registration number: NCT02474017.

A phosphatidylserine species inhibits a range of TLR- but not IL-1beta-induced inflammatory responses by disruption of membrane microdomains.

TLRs detect conserved molecular patterns that are unique to microbes, enabling tailored responses to invading pathogens and modulating a multitude of immunopathological conditions. We investigated the ability of a naturally occurring stearoyl-arachidonoyl form of phosphatidylserine (SAPS) to inhibit the proinflammatory effects of TLR agonists in models of inflammation investigating the interaction of leukocytes with epithelial and endothelial cells. The responses to LPS of both epithelial and endothelial cells were highly amplified in the presence of PBMCs. Coincubation with SAPS markedly inhibited activation of cocultures by LPS, principally through inhibition of the TLR4 signaling pathway in PBMCs; however, this was not through downmodulation of TLR4 or coreceptor expression, nor was IL-1beta-induced cytokine release affected. SAPS also impaired Pam(3)CSK(4) (TLR2/1), Gardiquimod (TLR7/8), and Streptococcus pneumoniae-induced cytokine release, but had only modest effects on poly(I:C) (TLR3)-induced responses. Fluorescence resonance energy transfer analysis of molecular associations revealed that SAPS disrupted the association of both TLR4 and TLR2 with their respective membrane partners that are required for signaling. Thus, our data reinforce the existence and importance of cooperative networks of TLRs, tissue cells, and leukocytes in mediating innate immunity, and identify a novel disrupter of membrane microdomains, revealing the dependence of TLR signaling on localization within these domains.

Equivalent Systemic Exposure to Fluticasone Propionate/Salmeterol Following Single Inhaled Doses from Advair Diskus and Wixela Inhub: Results of Three Pharmacokinetic Bioequivalence Studies.

Background: Wixela® Inhub® was developed to deliver inhaled fluticasone propionate/salmeterol (FP/S) combination as a substitutable generic equivalent to Advair® Diskus®. These studies aimed to confirm the pharmacokinetic bioequivalence (BE) of FP/S after single doses of Wixela Inhub (test [T]) and Advair Diskus (reference [R]). Methods: Three open-label, randomized, two-way crossover, single-dose studies in healthy subjects (N = 66 each) compared the systemic exposure of FP and salmeterol after inhalation from three dose strengths of FP/S (100/50, 250/50, or 500/50 µg) delivered from T and R. Primary BE endpoints were the area under the plasma concentration-time curve from time = 0 to the last measurable concentration (AUC(0-t)) and the maximum observed plasma concentration (Cmax) for both FP and S. The BE acceptance criteria specified that the 90% confidence intervals (CIs) of the geometric mean T/R ratios for AUC(0-t) and Cmax can be contained within 0.80-1.25 for both FP and salmeterol. Results: Wixela Inhub met the acceptance criteria for BE for FP and salmeterol at each dose strength. Estimated AUC(0-t) and Cmax geometric mean ratios (T/R [90% CI]) for FP were, respectively, 1.04 (1.00-1.08) and 0.92 (0.87-0.96) for 100/50 µg FP/S, 1.07 (1.02-1.13) and 1.01 (0.95-1.07) for 250/50 µg, and 0.97 (0.92, 1.00) and 0.90 (0.86-0.93) for 500/50 µg. Estimated AUC(0-t) and Cmax ratios for salmeterol were, respectively, 1.08 (1.04-1.11) and 1.00 (0.94-1.04) for 100/50 µg FP/S, 1.03 (0.99-1.07) and 0.93 (0.87-1.00) for 250/50 µg, and 1.00 (0.96-1.04) and 0.86 (0.81-0.91) for 500/50 µg. FP/S at all doses via both T and R was comparably well tolerated. Conclusions: Wixela Inhub was bioequivalent to Advair Diskus at all three dose strengths for both FP and S, providing direct evidence of equivalent systemic safety and indirect evidence for equivalent pulmonary deposition.

Wixela Inhub: Dosing Performance In Vitro and Inhaled Flow Rates in Healthy Subjects and Patients Compared with Advair Diskus.

Background: Wixela™ Inhub™ is a fluticasone propionate/salmeterol dry powder inhaler developed as a generic equivalent of Advair Diskus® for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Wixela Inhub and Advair Diskus are comparable in terms of functionality, user interface, and device resistance. The primary objectives of the studies were to evaluate in vitro dose delivery with Wixela Inhub compared with Advair Diskus at relevant flow rates and to explore inhalation profiles generated by patients with asthma or COPD. Methods:In vitro studies: Emitted dose (ED) and individual dose aerodynamic particle size distribution (APSD) were measured at flow rates ranging from 30 to 90 L min-1. Patient inhalation study: Inhalation profile recording was conducted three times in each patient (40 children with asthma, 14 adults with asthma, and 14 adults with severe-to-very-severe COPD) with an empty Inhub in an open-label study. The primary endpoint was peak inhaled flow rate (PIFR). An additional endpoint was peak pressure drop. Results:In vitro studies: ED and APSD delivered from Wixela Inhub showed low flow dependency across the patient-relevant flow-rate range. Wixela Inhub gave in vitro performance comparable with Advair Diskus for all strengths and flow rates. Patient inhalation study: For Inhub, mean PIFR was lowest for children with asthma ages 4 to 7 years (50.6 L min-1) and highest for adults with asthma (74.8 L min-1). For adults with severe-to-very-severe COPD, mean PIFR was 69.5 L min-1 with Inhub. The PIFRs observed with Diskus were higher than those with Inhub, consistent with slightly higher resistance measured in vitro. The difference in resistance did not impact demonstration of bioequivalence and does not impact substitutability of the product. Peak pressure drop values were comparable between Diskus and Inhub. Conclusions: Comparable in vitro performance of Wixela Inhub to Advair Diskus confirmed that Wixela Inhub is a generic equivalent to Advair Diskus across all patient groups.