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1.
J Orthop ; 22: 543-547, 2020.
Article in English | MEDLINE | ID: mdl-33208991

ABSTRACT

AIMS: There is very little published literature on Enhanced Recovery Principles (ERP) used in primary joint replacements applied to revision hip and knee arthroplasty (rTHA, rTKA). METHODS: Retrospective series of 268 rTHA and rTKA surgeries from 2010 -2018, treated with ERP, focusing on multimodal pain management, blood management and early functional recovery. RESULTS: No patients from the latest cohort required readmission within 6 weeks. Only 20 patients (7.5%) required a blood transfusion. Surgical site local anaesthetic infiltration was associated with lower PCA use in aseptic rTHA and rTKA (p<0.001; p<0.001). Revisions for infection had a longer length of stay (LOS) and increased PCA usage in both rTHA (6.5 vs. 5.2 days) and rTKA (10.1 vs. 5.3 days), similar to our previous study.1 Use of an intra-articular catheter for analgesia in rTKA showed reduced PCA use. Tourniquets were not beneficial for blood loss in rTKA and had greater PCA use post-operatively (p<0.001). CONCLUSION: The application of ERP to revision THA and TKA surgery is safe and effective.

2.
Am J Respir Cell Mol Biol ; 63(5): 591-600, 2020 11.
Article in English | MEDLINE | ID: mdl-32706623

ABSTRACT

Viral-induced exacerbation of asthma remains a major cause of hospitalization and mortality. New human-relevant models of the airways are urgently needed to understand how respiratory infections may trigger asthma attacks and to advance treatment development. Here, we describe a new human-relevant model of rhinovirus-induced asthma exacerbation that recapitulates viral infection of asthmatic airway epithelium and neutrophil transepithelial migration, and enables evaluation of immunomodulatory therapy. Specifically, a microengineered model of fully differentiated human mucociliary airway epithelium was stimulated with IL-13 to induce a T-helper cell type 2 asthmatic phenotype and infected with live human rhinovirus 16 (HRV16) to reproduce key features of viral-induced asthma exacerbation. We observed that the infection with HRV16 replicated key hallmarks of the cytopathology and inflammatory responses observed in human airways. Generation of a T-helper cell type 2 microenvironment through exogenous IL-13 stimulation induced features of asthmatic airways, including goblet cell hyperplasia, reduction of cilia beating frequency, and endothelial activation, but did not alter rhinovirus infectivity or replication. High-resolution kinetic analysis of secreted inflammatory markers revealed that IL-13 treatment altered IL-6, IFN-λ1, and CXCL10 secretion in response to HRV16. Neutrophil transepithelial migration was greatest when viral infection was combined with IL-13 treatment, whereas treatment with MK-7123, a CXCR2 antagonist, reduced neutrophil diapedesis in all conditions. In conclusion, our microengineered Airway Lung-Chip provides a novel human-relevant platform for exploring the complex mechanisms underlying viral-induced asthma exacerbation. Our data suggest that IL-13 may impair the hosts' ability to mount an appropriate and coordinated immune response to rhinovirus infection. We also show that the Airway Lung-Chip can be used to assess the efficacy of modulators of the immune response.


Subject(s)
Asthma/virology , Bioengineering , Disease Progression , Lab-On-A-Chip Devices , Lung/pathology , Lung/virology , Microtechnology , Models, Biological , Cell Movement , Cells, Cultured , Cytopathogenic Effect, Viral , Humans , Neutrophil Infiltration , Receptors, Interleukin-8B/antagonists & inhibitors , Receptors, Interleukin-8B/metabolism , Rhinovirus
3.
Integr Comp Biol ; 60(2): 440-455, 2020 08 01.
Article in English | MEDLINE | ID: mdl-32617553

ABSTRACT

Here we describe examples of studies that have contributed both to a basic understanding of the biology of imperiled marine turtles, and to their management and conservation. Key elements include, first and foremost, correctly identifying species that differ strikingly in their morphology at different life stages because with growth, they change size by several orders of magnitude and have accompanying shape changes. We also review comprehensive field studies documenting the need for management actions to correct abnormal shifts in sex ratios caused by climate change. We highlight the need to describe those perturbations in terms that are clear to regulators and personnel responsible for management and conservation policies. Finally, we review several basic studies that enhance our understanding of how selection has shaped morphological, functional, and performance attributes, and describe how that knowledge can be applied to the tasks required for enhancing species recovery.


Subject(s)
Conservation of Natural Resources , Life History Traits , Nesting Behavior , Turtles/anatomy & histology , Turtles/physiology , Animals , Climate Change , Female , Male
4.
J Aerosol Med Pulm Drug Deliv ; 32(5): 251-265, 2019 10.
Article in English | MEDLINE | ID: mdl-31084462

ABSTRACT

Background: A highly potent pan-Janus kinase (JAK) inhibitor with excellent kinome selectivity was developed for topical delivery to treat severe asthma. This poorly soluble drug discovery candidate, iJAK-001, is expected to exhibit long duration of JAK/STAT pathway inhibition at low doses in asthmatics because of depot effect after dry powder inhalation. Human dose projection for inhaled molecules with low aqueous solubility remains to be a daunting challenge because of several limitations: (1) bioanalytical measurement of dissolved fraction after inhalation of solid particles is uncertain; (2) distribution of these particles is not homogenous in the lung; (3) in vitro solubility measurements to estimate fraction dissolved may not be a reflection of local surface lung concentration; (4) lack of a surrogate biomarker of lung target engagement, and (5) invasive procedure needed to sample human lung tissue in the clinic. Methods: We leveraged in silico, in vitro, and in vivo tools preclinically and found significant differences in lung to plasma partition ratio when iJAK-001 was given intravenously (IV) or intratracheally in a solution-based formulation versus that in suspension, as well as pharmacodynamic response in preclinical asthma models when delivered systemically via IV infusion versus inhaled. Results and Conclusion: The combined results from above suggest that caution must be exercised using either lung or plasma exposure for human dose projection. Instead, using the local inhibitor concentration estimate based on delivery efficiency, dose, fraction absorbed, and rate of absorption normalized by lung (cardiac) blood flow may be more appropriate for dose projection.


Subject(s)
Asthma/drug therapy , Janus Kinase Inhibitors/administration & dosage , Lung/metabolism , Administration, Inhalation , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Dry Powder Inhalers , Humans , Janus Kinase Inhibitors/pharmacokinetics , Janus Kinase Inhibitors/pharmacology , Male , Rats , Rats, Inbred BN , Rats, Wistar , Sheep , Solubility , Tissue Distribution
5.
Innate Immun ; 25(2): 132-143, 2019 02.
Article in English | MEDLINE | ID: mdl-30774010

ABSTRACT

Crohn's disease (CD) is a chronic disorder of the gastrointestinal tract characterized by inflammation and intestinal epithelial injury. Loss of function mutations in the intracellular bacterial sensor NOD2 are major risk factors for the development of CD. In the absence of robust bacterial recognition by NOD2 an inflammatory cascade is initiated through alternative PRRs leading to CD. In the present study, MCC950, a specific small molecule inhibitor of NLR pyrin domain-containing protein 3 (NLRP3), abrogated dextran sodium sulfate (DSS)-induced intestinal inflammation in Nod2-/- mice. NLRP3 inflammasome formation was observed at a higher rate in NOD2-deficient small intestinal lamina propria cells after insult by DSS. NLRP3 complex formation led to an increase in IL-1ß secretion in both the small intestine and colon of Nod2ko mice. This increase in IL-1ß secretion in the intestine was attenuated by MCC950 leading to decreased disease severity in Nod2ko mice. Our work suggests that NLRP3 inflammasome activation may be a key driver of intestinal inflammation in the absence of functional NOD2. NLRP3 pathway inhibition can prevent intestinal inflammation in the absence of robust NOD2 signaling.


Subject(s)
Colitis/immunology , Crohn Disease/immunology , Gastrointestinal Microbiome/immunology , Inflammasomes/metabolism , Intestinal Mucosa/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Nod2 Signaling Adaptor Protein/genetics , Animals , Colitis/chemically induced , Dextran Sulfate , Disease Models, Animal , Furans/administration & dosage , Furans/pharmacology , Heterocyclic Compounds, 4 or More Rings , Humans , Indenes , Interleukin-1beta/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , Signal Transduction , Sulfonamides/administration & dosage , Sulfonamides/pharmacology , Sulfones
6.
Article in English | MEDLINE | ID: mdl-30367271

ABSTRACT

Hatchling marine turtles emerge at night from underground nests on oceanic beaches and then use visual cues to crawl from the nest site to the sea ("seafinding"). However, the light wavelengths (λ's) used to accomplish this orientation have not been thoroughly documented, nor do we understand why some λ's are favored over others. We measured nocturnal radiance on the horizon at 20 nm intervals between 340 and 600 nm at two nesting beach sites and then, under laboratory conditions, determined the lowest intensities of those λ's that induced green turtle and loggerhead hatchlings to crawl toward each light source (a low positive "phototaxis threshold"). Both species were similarly sensitive and were attracted to all λ's. Radiance measures at all λ's were greater toward the seaward horizon than toward the landward horizon, providing an important orientation cue regardless of variation in lunar illumination. Previous studies document that both species detect λ's longer than those that are most attractive. We hypothesize that seafinding is a specialized response mediated by cones that are sensitive to the shorter λ's (to minimize the effects of dark noise) but such as rods, are especially sensitive to low levels of nocturnal illumination.


Subject(s)
Light , Phototaxis , Spatial Navigation , Turtles , Animals , Cues , Lighting , Motor Activity , Nesting Behavior , Oceans and Seas , Species Specificity
7.
Pulm Pharmacol Ther ; 53: 12-19, 2018 12.
Article in English | MEDLINE | ID: mdl-30205157

ABSTRACT

BACKGROUND: Airway smooth muscle (ASM) contributes to local inflammation and plays an immunomodulatory role in airway diseases. This is partially regulated by p38 mitogen-activated protein kinase (MAPK), which further activates two closely related isoforms of the MAPK-activated protein kinases (MKs), MK2 and MK3. The MKs have similar substrate specificities but less is known about differences in their functional responses. This study was undertaken to identify differential downstream inflammatory targets of MK2 and MK3 signaling and assess cross-talk between the MAPK pathway and NF-κB signaling relevant to ASM function. METHODS: Wild-type and kinase-deficient MK2 (MK2WT, MK2KR) and MK3 (MK3WT, MK33A) were expressed in human ASM cells stimulated for 20 h with 10 ng/ml each interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Inflammatory mediator secretion was assessed by Luminex assays and ELISA. Signaling pathway activation was monitored by Western blotting. RESULTS: Expression of these MKs and stimulation with 10 ng/ml IL-1ß, TNFα and IFNγ for 20 h did not affect secretion of multiple cytokines including IL-4, IL-5, IL-13 and monocyte chemotactic protein (MCP)-1/CCL2 but did differentially affect the secretion of regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF). RANTES/CCL5 secretion was decreased by MK2WT or MK3WT and stimulated by inhibition of MK2 or MK3 activity with expression of the kinase-deficient enzymes MK2KR or MK33A. IL-6 and GM-CSF secretion was decreased by inhibition of MK2 activity with MK2KR and while MK3WT had no effect, the kinase-deficient MK33A further decreased secretion of these mediators. Cross-talk of the MKs with other signaling pathways was investigated by examining NF-κB activation, which was inhibited by expression of MK3 but not affected by MK2. CONCLUSIONS: These results suggest an inhibitory role for MK2 and MK3 activity in RANTES/CCL5 secretion and cross-talk of MK3 with NF-κB to regulate IL-6 and GM-CSF. These findings differentiate MK2 and MK3 function in ASM cells and provide insight that may enable selective targeting of MKs in ASM to modulate local inflammation in airway disease.


Subject(s)
Bronchi/cytology , Intracellular Signaling Peptides and Proteins/genetics , Myocytes, Smooth Muscle/metabolism , Protein Serine-Threonine Kinases/genetics , Blotting, Western , Cells, Cultured , Chemokine CCL5/metabolism , Chemokines/metabolism , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Inflammation/pathology , Inflammation Mediators/metabolism , Interleukin-6/metabolism , MAP Kinase Signaling System/physiology , Myocytes, Smooth Muscle/pathology , NF-kappa B/metabolism , Signal Transduction
8.
J Morphol ; 279(9): 1224-1233, 2018 09.
Article in English | MEDLINE | ID: mdl-30105840

ABSTRACT

In this study, we compare and contrast armoring strategies during early ontogeny among three related species of marine turtles: the hawksbill, a species that diverged about 29 mya from the loggerhead and Kemp's ridley, which diverged from one another about 16 mya. Our purpose was to determine whether there was a correlation between divergence time and the evolution of unique morphological armoring specializations among these species. To find out, we completed a more detailed analysis of shell morphology for all of the species that revealed the following patterns. First, each species has evolved a somewhat different armoring strategy, suggesting that shell morphological evolution is surprisingly flexible. Second, hawksbills possess armoring features that are unique among all marine turtle species, suggesting a correlation between divergence through time and divergence in morphology. However, hawksbills also frequent coral reefs and selection pressures promoting their survival in those habitats may also have shaped their unique morphology. In contrast, loggerhead and Kemp's ridley turtles share similar armoring features that differ primarily in when during ontogeny they appear and in their degree of expression. Third, the armoring adaptations shown generally by juvenile marine turtles resemble those found among marine fishes of comparable size, probably because both small turtles and fishes are exposed to similar predators that promote evolutionarily similar adaptations.


Subject(s)
Animal Shells/anatomy & histology , Phylogeny , Turtles/anatomy & histology , Analysis of Variance , Animals , Sample Size , Turtles/growth & development
9.
PLoS One ; 12(7): e0180870, 2017.
Article in English | MEDLINE | ID: mdl-28719615

ABSTRACT

While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the 'immune fingerprint' of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.


Subject(s)
Drug Evaluation, Preclinical/methods , Immune System/drug effects , Small Molecule Libraries/pharmacology , Chemokines/biosynthesis , Humans , Immune System/cytology , Immune System/immunology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Phagocytes/drug effects , Phagocytes/immunology , Phagocytes/metabolism , Reactive Oxygen Species/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Toll-Like Receptors/metabolism , Transcriptome/drug effects
10.
J Pharmacol Exp Ther ; 362(2): 338-346, 2017 08.
Article in English | MEDLINE | ID: mdl-28611093

ABSTRACT

CXC chemokine receptor 2 (CXCR2) is a key receptor in the chemotaxis of neutrophils to sites of inflammation. The studies reported here describe the pharmacological characterization of danirixin, a CXCR2 antagonist in the diaryl urea chemical class. Danirixin has high affinity for CXCR2, with a negative log of the 50% inhibitory concentration (pIC50) of 7.9 for binding to Chinese hamster ovary cell (CHO)-expressed human CXCR2, and 78-fold selectivity over binding to CHO-expressed CXCR1. Danirixin is a competitive antagonist against CXCL8 in Ca2+-mobilization assays, with a KB (the concentration of antagonist that binds 50% of the receptor population) of 6.5 nM and antagonist potency (pA2) of 8.44, and is fully reversible in washout experiments over 180 minutes. In rat and human whole-blood studies assessing neutrophil activation by surface CD11b expression following CXCL2 (rat) or CXCL1 (human) challenge, danirixin blocks the CD11b upregulation with pIC50s of 6.05 and 6.3, respectively. Danirixin dosed orally also blocked the influx of neutrophils into the lung in vivo in rats following aerosol lipopolysaccharide or ozone challenge, with median effective doses (ED50s) of 1.4 and 16 mg/kg respectively. Thus, danirixin would be expected to block chemotaxis in disease states in which neutrophils are increased in response to inflammation, such as pulmonary diseases. In comparison with navarixin, a CXCR2 antagonist from a different chemical class, the binding characterization of danirixin is distinct. These observations may offer insight into the previously observed clinical differences in induction of neutropenia between these compounds.


Subject(s)
Piperidines/pharmacology , Receptors, Interleukin-8B/antagonists & inhibitors , Receptors, Interleukin-8B/metabolism , Sulfones/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Humans , Interleukin-8/pharmacology , Male , Rats , Rats, Inbred Lew
11.
J Pharmacol Exp Ther ; 360(2): 324-340, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27927912

ABSTRACT

(R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl] carbamoyl}phenyl)sulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide trifluoroacetic acid (GS-5759) is a bifunctional ligand composed of a quinolinone-containing pharmacophore [ß2-adrenoceptor agonist orthostere (ß2A)] found in several ß2-adrenoceptor agonists, including indacaterol, linked covalently to a phosphodiesterase 4 (PDE4) inhibitor related to 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) by a pent-1-yn-1-ylbenzene spacer. GS-5759 had a similar affinity for PDE4B1 and the native ß2-adrenoceptor expressed on BEAS-2B human airway epithelial cells. However, compared with the monofunctional parent compound, ß2A, the KA of GS-5759 for the ß2-adrenoceptor was 35-fold lower. Schild analysis determined that the affinities of the ß-adrenoceptor antagonists, (2R,3R)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol (ICI 118551) and propranolol, were agonist-dependent, being significantly lower for GS-5759 than ß2A. Collectively, these data can be explained by "forced proximity," bivalent binding where the pharmacophore in GS-5759 responsible for PDE4 inhibition also interacts with a nonallosteric domain within the ß2-adrenoceptor that enhances the affinity of ß2A for the orthosteric site. Microarray analyses revealed that, after 2-hour exposure, GS-5759 increased the expression of >3500 genes in BEAS-2B cells that were highly rank-order correlated with gene expression changes produced by indacaterol and GSK 256066 in combination (Ind/GSK). Moreover, the line of regression began close to the origin with a slope of 0.88, indicating that the magnitude of most gene expression changes produced by Ind/GSK was quantitatively replicated by GS-5759. Thus, GS-5759 is a novel compound exhibiting dual ß2-adrenoceptor agonism and PDE4 inhibition with potential to interact on target tissues in a synergistic manner. Such polypharmacological behavior may be particularly effective in chronic obstructive pulmonary disease and other complex disorders where multiple processes interact to promote disease pathogenesis and progression.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Epithelial Cells/drug effects , Gene Expression Regulation/drug effects , Pulmonary Disease, Chronic Obstructive/genetics , Quinolones/pharmacology , Receptors, Adrenergic, beta-2/metabolism , Respiratory System/pathology , Sulfones/pharmacology , Adrenergic beta-2 Receptor Agonists/pharmacology , Adrenergic beta-2 Receptor Agonists/therapeutic use , Aminoquinolines/pharmacology , Cell Line , Drug Interactions , Epithelial Cells/metabolism , Humans , Indans/pharmacology , Phosphodiesterase 4 Inhibitors/pharmacology , Phosphodiesterase 4 Inhibitors/therapeutic use , Pulmonary Disease, Chronic Obstructive/drug therapy , Pulmonary Disease, Chronic Obstructive/pathology , Quinolones/therapeutic use , Sulfones/therapeutic use
12.
J Aerosol Med Pulm Drug Deliv ; 29(4): 362-77, 2016 08.
Article in English | MEDLINE | ID: mdl-26859446

ABSTRACT

BACKGROUND: Understanding the relationship between dose, lung exposure, and drug efficacy continues to be a challenging aspect of inhaled drug development. An experimental inhalation platform was developed using mometasone furoate to link rodent lung exposure to its in vivo pharmacodynamic (PD) effects. METHODS: We assessed the effect of mometasone delivered directly to the lung in two different rodent PD models of lung inflammation. The data obtained were used to develop and evaluate a mathematical model to estimate drug dissolution, transport, distribution, and efficacy, following inhaled delivery in rodents and humans. RESULTS: Mometasone directly delivered to the lung, in both LPS and Alternaria alternata rat models, resulted in dose dependent inhibition of BALf cellular inflammation. The parameters for our mathematical model were calibrated to describe the observed lung and systemic exposure profiles of mometasone in humans and in animal models. We found that physicochemical properties, such as lung fluid solubility and lipophilicity, strongly influenced compound distribution and lung retention. CONCLUSIONS: Presently, we report on a novel and sophisticated mathematical model leading to improvements in a current inhaled drug development practices by providing a quantitative understanding of the relationship between PD effects and drug concentration in lungs.


Subject(s)
Alternariosis/drug therapy , Anti-Inflammatory Agents/administration & dosage , Drug Dosage Calculations , Lung Diseases, Fungal/drug therapy , Lung/drug effects , Models, Biological , Mometasone Furoate/administration & dosage , Pneumonia/drug therapy , Administration, Inhalation , Aerosols , Alternaria , Alternariosis/metabolism , Alternariosis/microbiology , Alternariosis/physiopathology , Animals , Anti-Inflammatory Agents/pharmacokinetics , Disease Models, Animal , Humans , Lipopolysaccharides , Lung/metabolism , Lung/physiopathology , Lung Diseases, Fungal/metabolism , Lung Diseases, Fungal/microbiology , Lung Diseases, Fungal/physiopathology , Male , Mometasone Furoate/pharmacokinetics , Pneumonia/chemically induced , Pneumonia/metabolism , Pneumonia/physiopathology , Rats, Inbred BN , Rats, Sprague-Dawley , Species Specificity , Tissue Distribution
13.
Nat Methods ; 13(2): 151-7, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26689262

ABSTRACT

Here we describe the development of a human lung 'small airway-on-a-chip' containing a differentiated, mucociliary bronchiolar epithelium and an underlying microvascular endothelium that experiences fluid flow, which allows for analysis of organ-level lung pathophysiology in vitro. Exposure of the epithelium to interleukin-13 (IL-13) reconstituted the goblet cell hyperplasia, cytokine hypersecretion and decreased ciliary function of asthmatics. Small airway chips lined with epithelial cells from individuals with chronic obstructive pulmonary disease recapitulated features of the disease such as selective cytokine hypersecretion, increased neutrophil recruitment and clinical exacerbation by exposure to viral and bacterial infections. With this robust in vitro method for modeling human lung inflammatory disorders, it is possible to detect synergistic effects of lung endothelium and epithelium on cytokine secretion, identify new biomarkers of disease exacerbation and measure responses to anti-inflammatory compounds that inhibit cytokine-induced recruitment of circulating neutrophils under flow.


Subject(s)
Epithelium/drug effects , Inflammation/metabolism , Interleukin-13/pharmacology , Lab-On-A-Chip Devices , Lung Diseases/drug therapy , Lung Diseases/metabolism , Humans , Inflammation/pathology , Tissue Culture Techniques
14.
J Morphol ; 276(8): 929-40, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26126953

ABSTRACT

Marine turtles are large reptiles that compensate for high juvenile mortality by producing hundreds of hatchlings during a long reproductive lifespan. Most hatchlings are taken by predators during their migration to, and while resident in, the open ocean. Their survival depends upon crypticity, minimizing movement to avoid detection, and foraging efficiently to grow to a size too difficult for predators to either handle or swallow. While these behavioral antipredator tactics are known, changes in morphology accompanying growth may also improve survival prospects. These have been only superficially described in the literature. Here, we compare the similarities and differences in presumed morphological defenses of growing loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempii) posthatchlings, related species that differ in growth rate, timing of habitat shift (the return from oceanic to neritic locations), and size at maturity. In both species, vertebral spination and carapace widening increase disproportionally as small turtles grow, but later in ontogeny, the spines regress, sooner in ridley than in loggerhead turtles. Carapace widening occurs in both species but loggerheads are always longer than they are wide whereas in Kemp's ridley turtles, the carapace becomes as wide as long. Our analysis indicates that these changes are unrelated to when each species shifts habitat but are related to turtle size. We hypothesize that the spines function in small turtles as an early defense against gape-limited predators, but changes in body shape function throughout ontogeny-initially to make small turtles too wide to swallow and later by presenting an almost flat and hardened surface that large predators (such as a sharks) are unable to grasp. The extremely wide carapace of the Kemp's ridley may compensate for its smaller adult size (and presumed greater vulnerability) than the loggerhead.


Subject(s)
Turtles/anatomy & histology , Adaptation, Physiological , Animals , Turtles/physiology
15.
Pharmacol Res Perspect ; 2(4): e00046, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25505595

ABSTRACT

Bronchodilators are a central therapy for symptom relief in respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma, with inhaled ß 2-adrenoceptor agonists and anticholinergics being the primary treatments available. The present studies evaluated the in vivo pharmacology of (R)-6-[[3-[[4-[5-[[2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino]pent-1-ynyl]phenyl]carbamoyl]phenyl]sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GS-5759), a novel bifunctional compound with both phosphodiesterase 4 (PDE4) inhibitor and long-acting ß 2-adrenoceptor agonist (LABA) activity, which has been optimized for inhalation delivery. GS-5759 dose-dependently inhibited pulmonary neutrophilia in a lipopolysaccharide (LPS) aerosol challenge model of inflammation in rats with an ED50 ≤ 10 µg/kg. GS-5759 was also a potent bronchodilator with an ED50 of 0.09 µg/kg in guinea pigs and 3.4 µg/kg in dogs after methylcholine (MCh) and ragweed challenges respectively. In cynomolgus monkeys, GS-5759 was dosed as a fine-particle dry powder and was efficacious in the same dose range in both MCh and LPS challenge models, with an ED50 = 70 µg/kg for bronchodilation and ED50 = 4.9 µg/kg for inhibition of LPS-induced pulmonary neutrophilia. In models to determine therapeutic index (T.I.), efficacy for bronchodilation was evaluated against increased heart rate and GS-5759 had a T.I. of 700 in guinea pigs and >31 in dogs. In a ferret model of emesis, no emesis was seen at doses several orders of magnitude greater than the ED50 observed in the rat LPS inflammation model. GS-5759 is a bifunctional molecule developed for the treatment of COPD, which has both bronchodilator and anti-inflammatory activity and has the potential for combination as a triple therapy with a second compound, within a single inhalation device.

16.
Eur J Pharmacol ; 743: 106-16, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25261040

ABSTRACT

Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view of the clinical relationship between A. alternata and asthma, we sought to investigate the allergic activity of this antigen after direct application to the lungs of Brown Norway rats. Here we demonstrate that a single intratracheal instillation of A. alternata induces dose and time dependent eosinophil influx, edema and Type 2 helper cell cytokine production in the lungs of BN rats. We established the temporal profile of eosinophilic infiltration and cytokine production, such as Interleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable to Ovalbumin induced models of asthma and resulted in peak inflammatory responses 48h following a single challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascular and peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternata inflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptor-homologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreased eosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore, therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influx and Th2 cytokine production. Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechanisms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats is a valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors of allergic pulmonary inflammation.


Subject(s)
Alternaria/drug effects , Anti-Inflammatory Agents/pharmacology , Carbolines/pharmacology , Pneumonia/drug therapy , Receptors, Formyl Peptide/metabolism , Th2 Cells/drug effects , Allergens/immunology , Alternaria/immunology , Animals , Asthma/drug therapy , Asthma/immunology , Asthma/metabolism , Cytokines/immunology , Cytokines/metabolism , Eosinophils/drug effects , Eosinophils/immunology , Eosinophils/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Interleukin-13/immunology , Interleukin-13/metabolism , Interleukin-5/immunology , Interleukin-5/metabolism , Lung/drug effects , Lung/immunology , Lung/metabolism , Male , Ovalbumin/immunology , Ovalbumin/pharmacology , Pneumonia/immunology , Pneumonia/metabolism , Rats , Rats, Inbred BN , Receptors, Formyl Peptide/immunology , Th2 Cells/immunology
17.
Zoology (Jena) ; 117(2): 131-8, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24629459

ABSTRACT

Female marine turtles produce hundreds of offspring during their lifetime but few survive because small turtles have limited defenses and are vulnerable to many predators. Little is known about how small turtles improve their survival probabilities with growth though it is assumed that they do. We reared green turtles (Chelonia mydas) and loggerheads (Caretta caretta) from hatchlings to 13 weeks of age and documented that they grew wider faster than they grew longer. This pattern of allometric growth might enable small turtles to more quickly achieve protection from gape-limited predators, such as the dolphinfish (Coryphaena hippurus). As a test of that hypothesis, we measured how dolphinfish gape increased with length, reviewed the literature to determine how dolphinfish populations were size/age structured in nearby waters, and then determined the probability that a small turtle would encounter a fish large enough to consume it if it grew by allometry vs. by isometry (in which case it retained its hatchling proportions). Allometric growth more quickly reduced the probability of a lethal encounter than did isometric growth. On that basis, we suggest that allometry during early ontogeny may have evolved because it provides a survival benefit for small turtles.


Subject(s)
Adaptation, Physiological , Food Chain , Turtles/growth & development , Age Factors , Animals , Fishes/anatomy & histology , Mouth/anatomy & histology , Population Density
18.
J Pharmacol Exp Ther ; 349(1): 85-93, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24513870

ABSTRACT

Inhaled long-acting ß(2)-adrenoceptor agonists (LABA) that act as bronchodilators and the oral anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor roflumilast are both approved therapies for chronic obstructive pulmonary disease (COPD). Here we describe the activity of a novel, inhaled, bifunctional, small molecule (R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl]carbamoyl}phenyl)sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GS-5759), which has specific ß(2) agonist and PDE4 inhibitory activity. GS-5759 demonstrated potent and full agonist activity at ß(2) adrenoceptors (EC(50) = 8 ± 4 nM) and is a potent inhibitor of the PDE4 enzyme (IC(50) = 5 ± 3 nM). In cell assays, GS-5759 inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) production in human peripheral mononuclear cells (PBMC) with an IC(50) = 0.3 nM [confidence interval (CI) 0.1-0.6] and in human neutrophils formyl-methionyl-leucyl-phenylalanine (fMLP)-induced super oxide anion production with an IC(50) = 3 nM (CI 0.8-8). The addition of the ß(2) antagonist ICI 118551 shifted the IC(50) in these cell assays to 4 and 38 nM, respectively, demonstrating the contribution of both ß(2) agonist and PDE4 inhibitory activity to GS-5759. GS-5759 was also a potent inhibitor of profibrotic and proinflammatory mediator release from human lung fibroblasts. GS-5759 relaxed guinea pig airway smooth muscle strips precontracted with carbachol in a concentration-dependent manner with an EC(50) = 0.5 µM (CI 0.2-2) and had slow dissociation kinetics with an Off T(1/2) > 720 minutes at an EC(80) concentration of 3 µM. GS-5759 is a novel bifunctional molecule with both potent ß(2) agonist and PDE4 inhibitor activity that could provide inhaled bronchodilator and anti-inflammatory therapy for COPD.


Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Fibroblasts/drug effects , Leukocytes, Mononuclear/drug effects , Muscle, Smooth/drug effects , Phosphodiesterase 4 Inhibitors/pharmacology , Quinolones/pharmacology , Respiratory System/drug effects , Sulfones/pharmacology , Adrenergic beta-2 Receptor Agonists/chemical synthesis , Adrenergic beta-2 Receptor Agonists/chemistry , Animals , Cell Culture Techniques , Cytokines/antagonists & inhibitors , Cytokines/immunology , Fibroblasts/enzymology , Fibroblasts/immunology , Fibroblasts/metabolism , Guinea Pigs , Humans , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Male , Molecular Structure , Muscle, Smooth/enzymology , Muscle, Smooth/immunology , Muscle, Smooth/metabolism , Phosphodiesterase 4 Inhibitors/chemical synthesis , Phosphodiesterase 4 Inhibitors/chemistry , Pulmonary Disease, Chronic Obstructive/drug therapy , Quinolones/chemical synthesis , Quinolones/chemistry , Respiratory System/enzymology , Respiratory System/immunology , Respiratory System/metabolism , Sulfones/chemical synthesis , Sulfones/chemistry , Time Factors
19.
J Pharmacol Exp Ther ; 345(2): 260-70, 2013 May.
Article in English | MEDLINE | ID: mdl-23435542

ABSTRACT

Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.2]octane; umeclidinium). The affinity (Ki) of GSK573719 for the cloned human M1-M5 mAChRs ranged from 0.05 to 0.16 nM. Dissociation of [(3)H]GSK573719 from the M3 mAChR was slower than that for the M2 mAChR [half-life (t1/2) values: 82 and 9 minutes, respectively]. In Chinese hamster ovary cells transfected with recombinant human M3 mAChRs, GSK573719 demonstrated picomolar potency (-log pA2 = 23.9 pM) in an acetylcholine (Ach)-mediated Ca(2+) mobilization assay. Concentration-response curves indicate competitive antagonism with partial reversibility after drug washout. Using isolated human bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA2 = 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED50 value was 0.02 µg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 µg elicited 50% bronchoprotection for >24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.


Subject(s)
Lung Diseases/drug therapy , Muscarinic Antagonists/therapeutic use , Quinuclidines/therapeutic use , Administration, Inhalation , Animals , CHO Cells , Calcium/metabolism , Carbachol/pharmacology , Cholinergic Antagonists/pharmacology , Cricetinae , Cricetulus , Guinea Pigs , Kinetics , Lung/drug effects , Mice , Mice, Inbred BALB C , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/administration & dosage , Plethysmography , Quinuclidines/administration & dosage , Receptor, Muscarinic M3/drug effects , Receptors, Muscarinic , Scopolamine Derivatives/pharmacology , Tiotropium Bromide
20.
Bioorg Med Chem Lett ; 22(23): 7087-91, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-23099092

ABSTRACT

Tyrosine ureas had been identified as potent muscarinic receptor antagonists with promising in vivo activity. Controlling the stereochemistry of the chiral quaternary ammonium center had proved to be a serious issue for this series, however. Herein we describe the preparation and SAR of tyrosine urea antagonists containing achiral quaternary ammonium centers. The most successful such moiety was the 2-methylimidazo[2,1-b][1,3]thiazol-7-ium group which yielded highly potent antagonists with long duration of action in an inhaled animal model of bronchoconstriction.


Subject(s)
Muscarinic Antagonists/chemistry , Quaternary Ammonium Compounds/chemistry , Receptors, Muscarinic/chemistry , Tyrosine/chemistry , Urea/analogs & derivatives , Animals , Bronchi/drug effects , Mice , Muscarinic Antagonists/chemical synthesis , Muscarinic Antagonists/pharmacology , Receptors, Muscarinic/metabolism , Structure-Activity Relationship , Urea/chemical synthesis , Urea/pharmacology
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