The role of Wnt signaling in skin fibrosis.

Skin fibrosis is the excessive deposition of extracellular matrix in the dermis. Cutaneous fibrosis can occur following tissue injury, including burns, trauma, and surgery, resulting in scars that are disfiguring, limit movement and cause significant psychological distress for patients. Many molecular pathways have been implicated in the development of skin fibrosis, yet effective treatments to prevent or reverse scarring are unknown. The Wnt signaling pathways are known to play an important role in skin homeostasis, skin injury, and in the development of fibrotic skin diseases. This review provides a detailed overview of the role of the canonical Wnt signaling pathways in regulating skin scarring. We also discuss how Wnt signaling interacts with other known fibrotic molecular pathways to cause skin fibrosis. We further provide a summary of the different Wnt inhibitor types available for treating skin scarring. Understanding the role of the Wnt pathway in cutaneous fibrosis will accelerate the development of effective Wnt modulators for the treatment of skin fibrosis.

A Design of Experiment (DoE) approach to optimise spray drying process conditions for the production of trehalose/leucine formulations with application in pulmonary delivery.

The present study evaluates the effect of L-leucine concentration and operating parameters of a laboratory spray dryer on characteristics of trehalose dry powders, with the goal of optimizing production of these powders for inhaled drug delivery. Trehalose/L-leucine mixtures were spray dried from aqueous solution using a laboratory spray dryer. A factorial design of experiment (DoE) was undertaken and process parameters adjusted were: inlet temperature, gas flow rate, feed solution flow rate (pump setting), aspiration setting and L-leucine concentration. Resulting powders were characterised in terms of particle size, yield, residual moisture content, and glass transition temperature. Particle size was mainly influenced by gas flow rate, whereas product yield and residual moisture content were found to be primarily affected by inlet temperature and spray solution feed rate respectively. Interactions between a number of different process parameters were elucidated, as were relationships between different responses. The leucine mass ratio influenced the physical stability of powders against environmental humidity, and a high leucine concentration (30% w/w) protected amorphous trehalose from moisture induced crystallization. High weight ratio of leucine in the formulation, however, negatively impacted the aerosol performance. Thus, in terms of L-leucine inclusion in a formulation designed for pulmonary delivery, a balance needs to be found between physical stability and deposition characteristics.

Corticosteroid and long-acting ß-agonist therapy reduces epithelial goblet cell metaplasia.

BACKGROUND: Bronchial epithelial goblet cell metaplasia (GCM) with hyperplasia is a prominent feature of asthma, but the effects of treatment with corticosteroids alone or in combination with a long-acting ß2 -adrenergic receptor agonist (LABA) on GCM in the bronchial epithelium are unknown. OBJECTIVES: To determine whether corticosteroid alone or in combination with a LABA alters protein and gene expression pathways associated with IL-13-induced goblet cell metaplasia. RESULTS: We evaluated the effects of fluticasone propionate (FP) and of salmeterol (SM), on the response of well-differentiated cultured bronchial epithelial cells to interleukin-13 (IL-13). Outcome measures included gene expression of SPDEF/FOXa2, gene expression and protein production of MUC5AC/MUC5B and morphologic appearance of cultured epithelial cell sheets. We additionally analysed expression of these genes in bronchial epithelial brushings from healthy, steroid-naïve asthmatic and steroid-treated asthmatic subjects. In cultured airway epithelial cells, FP treatment inhibited IL-13-induced suppression of FOXa2 gene expression and up-regulation of SPDEF, alterations in gene and protein measures of MUC5AC and MUC5B and induction of GCM. The addition of SM synergistically modified the effects of FP modestly-only for gel-forming mucin MUC5AC. In bronchial epithelial cells recovered from asthmatic vs healthy human subjects, we found FOXa2 and MUC5B gene expression to be reduced and SPDEF and MUC5AC gene expression to be increased; these alterations were not observed in bronchial epithelial cells recovered after treatment with inhaled corticosteroids. CONCLUSION AND CLINICAL RELEVANCE: Corticosteroid treatment inhibits IL-13-induced GCM of the airways in asthma, possibly through its effects on SPDEF and FOXa2 regulation of mucin gene expression. These effects are modestly augmented by the addition of a long-acting ß-agonist. As we found evidence for drug treatment counteracting the effects of IL-13 on the epithelium, we conclude that further exploration into the mechanisms by which corticosteroids and long-acting ß2 -adrenergic agonists confer protection against pathologic airway changes is warranted.

Phenotype standardization for statin-induced myotoxicity.

Statins are widely used lipid-lowering drugs that are effective in reducing cardiovascular disease risk. Although they are generally well tolerated, they can cause muscle toxicity, which can lead to severe rhabdomyolysis. Research in this area has been hampered to some extent by the lack of standardized nomenclature and phenotypic definitions. We have used numerical and descriptive classifications and developed an algorithm to define statin-related myotoxicity phenotypes, including myalgia, myopathy, rhabdomyolysis, and necrotizing autoimmune myopathy.

A protective role for periostin and TGF-ß in IgE-mediated allergy and airway hyperresponsiveness.

BACKGROUND: The pathophysiology of asthma involves allergic inflammation and remodelling in the airway and airway hyperresponsiveness (AHR) to cholinergic stimuli, but many details of the specific underlying cellular and molecular mechanisms remain unknown. Periostin is a matricellular protein with roles in tissue repair following injury in both the skin and heart. It has recently been shown to be up-regulated in the airway epithelium of asthmatics and to increase active TGF-ß. Though one might expect periostin to play a deleterious role in asthma pathogenesis, to date its biological role in the airway is unknown. OBJECTIVE: To determine the effect of periostin deficiency on airway responses to inhaled allergen. METHODS: In vivo measures of airway responsiveness, inflammation, and remodelling were made in periostin deficient mice and wild-type controls following repeated intranasal challenge with Aspergillus fumigatus antigen. In vitro studies of the effects of epithelial cell-derived periostin on murine T cells were also performed. RESULTS: Surprisingly, compared with wild-type controls, periostin deficient mice developed increased AHR and serum IgE levels following allergen challenge without differences in two outcomes of airway remodelling (mucus metaplasia and peribronchial fibrosis). These changes were associated with decreased expression of TGF-ß1 and Foxp3 in the lungs of periostin deficient mice. Airway epithelial cell-derived periostin-induced conversion of CD4(+) CD25(-) cells into CD25(+) , Foxp3(+) T cells in vitro in a TGF-ß dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Allergen-induced increases in serum IgE and bronchial hyperresponsiveness are exaggerated in periostin deficient mice challenged with inhaled aeroallergen. The mechanism of periostin's effect as a brake on allergen-induced responses may involve augmentation of TGF-ß-induced T regulatory cell differentiation.

Genetic association between human chitinases and lung function in COPD.

Two primary chitinases have been identified in humans--acid mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Mammalian chitinases have been observed to affect the host's immune response. The aim of this study was to test for association between genetic variation in the chitinases and phenotypes related to chronic obstructive pulmonary disease (COPD). Polymorphisms in the chitinase genes were selected based on previous associations with respiratory diseases. Polymorphisms that were associated with lung function level or rate of decline in the Lung Health Study (LHS) cohort were analyzed for association with COPD affection status in four other COPD case-control populations. Chitinase activity and protein levels were also related to genotypes. In the caucasian LHS population, the baseline forced expiratory volume in one second (FEV(1)) was significantly different between the AA and GG genotypic groups of the AMCase rs3818822 polymorphism. Subjects with the GG genotype had higher AMCase protein and chitinase activity compared with AA homozygotes. For CHIT1 rs2494303, a significant association was observed between rate of decline in FEV(1) and the different genotypes. In the African American LHS population, CHIT1 rs2494303 and AMCase G339T genotypes were associated with rate of decline in FEV(1). Although a significant effect of chitinase gene alleles was found on lung function level and decline in the LHS, we were unable to replicate the associations with COPD affection status in the other COPD study groups.

Negative methacholine challenge tests in subjects who report physician-diagnosed asthma.

BACKGROUND: The frequency of adults reporting a history of asthma is rising. However, it is unclear whether this increased prevalence accurately demonstrates a rising trend or if it reflects an overall increase in asthma awareness. OBJECTIVE: To determine the frequency of negative methacholine bronchoprovocation tests in adults who report physician-diagnosed asthma and to explore the clinical characteristics of subjects with negative tests. METHODS: Data from methacholine challenge, spirometry, and physician assessment were analysed from 304 adults who reported physician-diagnosed asthma and responded to community-based advertising for asthma research studies. The clinical characteristics of methacholine-positive and -negative subjects were compared and a predictive model was tested to identify those characteristics associated with a negative test. RESULTS: Of the 304 subjects tested, 83 (27%) had a negative methacholine test. A negative test was positively associated with an adult-onset of symptoms (P<0.001), normal forced expiratory volume in 1 s (P<0.001), and having no history of exacerbation requiring oral steroids (P=0.03). Over half (60%) of those with a negative test reported weekly asthma-like symptoms (cough, dyspnoea, chest tightness, or wheeze), while 39% reported emergency department visits for asthma-like symptoms. CONCLUSIONS AND CLINICAL RELEVANCE: A sizeable percentage of subjects who report physician-diagnosed asthma have a negative methacholine challenge test. These subjects are characterized by diagnosis of asthma as an adult and by normal or near normal spirometry. Caution should be exercised in the assessment and diagnosis of adults presenting with asthma-like symptoms, because they may not have asthma. Further diagnostic studies, including bronchoprovocation testing, are warranted in this patient group, especially if their spirometry is normal.

Acute exacerbations of asthma: epidemiology, biology and the exacerbation-prone phenotype.

Asthma is a highly prevalent chronic respiratory disease affecting 300 million people world-wide. A significant fraction of the cost and morbidity of asthma derives from acute care for asthma exacerbations. In the United States alone, there are approximately 15 million outpatient visits, 2 million emergency room visits, and 500,000 hospitalizations each year for management of acute asthma. Common respiratory viruses, especially rhinoviruses, cause the majority of exacerbations in children and adults. Infection of airway epithelial cells with rhinovirus causes the release of pro-inflammatory cytokines and chemokines, as well as recruitment of inflammatory cells, particularly neutrophils, lymphocytes, and eosinophils. The host response to viral infection is likely to influence susceptibility to asthma exacerbation. Having had at least one exacerbation is an important risk factor for recurrent exacerbations suggesting an 'exacerbation-prone' subset of asthmatics. Factors underlying the 'exacerbation-prone' phenotype are incompletely understood but include extrinsic factors: cigarette smoking, medication non-compliance, psychosocial factors, and co-morbidities such as gastroesophageal reflux disease, rhinosinusitis, obesity, and intolerance to non-steroidal anti-inflammatory medications; as well as intrinsic factors such as deficient epithelial cell production of the anti-viral type I interferons (IFN-alpha and IFN-beta). A better understanding of the biologic mechanisms of host susceptibility to recurrent exacerbations will be important for developing more effective preventions and treatments aimed at reducing the significant cost and morbidity associated with this important global health problem.

Predicting worsening asthma control following the common cold.

The asthmatic response to the common cold is highly variable, and early characteristics that predict worsening of asthma control following a cold have not been identified. In this prospective multicentric cohort study of 413 adult subjects with asthma, the mini-Asthma Control Questionnaire (mini-ACQ) was used to quantify changes in asthma control and the Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) to measure cold severity. Univariate and multivariable models were used to examine demographic, physiological, serological and cold-related characteristics for their relationship to changes in asthma control following a cold. Clinically significant worsening of asthma control was observed following a cold (mean+/-SD increase in mini-ACQ score of 0.69+/-0.93). Univariate analysis demonstrated that season, centre location, cold duration and cold severity measurements were all associated with a change in asthma control. Multivariable analysis of the covariates available within the first 2 days of cold onset revealed that the day 2 and cumulative sum of day 1 and 2 WURSS-21 scores were significant predictors of the subsequent changes in asthma control. In asthmatic subjects, cold severity within the first 2 days can be used to predict subsequent changes in asthma control. This information may help clinicians prevent deterioration in asthma control following a cold.

Metabolism pathway of vinorelbine (Navelbine) in human: characterisation of the metabolites by HPLC-MS/MS.

The biotransformation of vinorelbine (VRL), an anti-neoplastic vinca-alkaloid derivate already marketed for nonsmall cell lung cancer and advanced breast cancer as an i.v. form and currently registered in several countries as an oral form, was investigated in human. Biological specimen from several human sources constituted the material for the metabolic identification in human. An isocratic liquid chromatographic system composed of 40 mM ammonium acetate (pH 3) and acetonitrile was used for separation of the potential metabolites of VRL. Tandem mass spectrometry with positive electrospray ionisation was used to enable the structural identification of the metabolites. A total of 17 metabolites (12 directly obtained from VRL and 5 involving sequential step pathways) were characterised with proposed structures for most of the metabolites. All metabolites went through phase I reactions by the way of deacetylation, dealkylation, oxidation and hydroxylation. No conjugates were observed. Despite the high number of metabolites quantified, VRL was the major compound observed whatever the matrix. Most of the metabolites rapidly disappeared from blood, except 4-O-deacetyl vinorelbine which was slowly cleared. Most of the enzymatic pathways involved in the metabolites strongly suggested the major role of cytochrome P450 in the biotransformation of vinorelbine.

Effect of a single dose of the selectin inhibitor TBC1269 on early and late asthmatic responses.

BACKGROUND: Selectins participate in the initial phase of leucocyte migration from circulation to inflamed tissues and may play a role in inflammatory cellular influx into airways in asthma. In the sheep asthma model, TBC1269, a pan-selectin antagonist, reduced late allergen response by 74%. OBJECTIVE: To determine whether a single dose of TBC1269 inhibits early (EAR) and late (LAR) asthmatic responses, and whether it inhibits sputum leucocyte influx after inhalation allergen challenge in atopic asthmatic subjects treated with bronchodilators only. METHODS: Twenty-one asthmatic subjects (mean+/-SD, age=32.5+/-6.7 years, 8 males, FEV1 percent predicted=84+/-15%) with known late asthmatic response based on a screening inhalation allergen challenge were randomly assigned to receive intravenous treatment with either placebo (n=11) or TBC1269 (n=10, 30 mg/kg) infused over 15 min immediately prior to a second (post-treatment) allergen challenge at least 4 weeks after the screening challenge. After each challenge, EAR and LAR were monitored for 7 h. In addition, sputum was induced 1 day before and 1 day after each allergen challenge. RESULTS: TBC1269 did not attenuate the EAR compared with placebo (largest fall in FEV1 within 1 h of 34.1+/-13.9% vs. 31.8+/-12.2% for TBC1269 and placebo groups respectively, P=0.61) or the LAR (largest fall in FEV1 between 3 and 7 h of 39.3+/-15.3% vs. 32.6+/-13.8%, P=0.24). TBC1269 had only minor effects on allergen-induced sputum eosinophilia. CONCLUSION: We conclude that TBC1269 administered before allergen challenge as a single intravenous dose does not attenuate early or late asthmatic responses to allergen in asthmatic subjects.

Remodeling of the airway epithelium in asthma.

Several pathologic changes occur in the airway epithelium in asthma, but the relationship between these changes and the initiation and progression of asthma remains poorly understood. One possibility is that changes in the structure and function of the epithelium induced by environmental exposure in genetically susceptible subjects represent primary pivotal events that occur early in the pathogenesis of asthma. Alternatively, these epithelial changes may occur simply as a consequence of pivotal early events in other systems, such as immune deviation in childhood to a helper T cell type 2 (Th2) subtype of CD4(+) cells. Epithelial desquamation in asthma represents a pathologic change that is frequently cited as important for the mechanisms of airway remodeling and airway hyperresponsiveness. Desquamation of the epithelium may not represent true pathology, however, but may instead be an artifact of tissue sampling and handling. Evidence is more firm for other pathologic changes in the epithelium. For example, goblet cell numbers are increased in asthma, leading to increases in stored mucins in the epithelium and in secreted mucins in sputum. The functional consequences of these changes include sputum production and airway narrowing, which lead to asthma exacerbations. Currently available data suggest that an important mechanism for goblet cell hyperplasia in asthma is the action of Th2 cytokines. Improved understanding of epithelial goblet cell abnormalities in asthma will hopefully lead to novel therapies for mucin hypersecretion, which is an important cause of morbidity and mortality.

Quality control within the Asthma Clinical Research Network.

Quality control and assurance are very important issues for multicenter clinical investigation. Although such investigation usually states that those factors are part of the investigation, at best this occurs to a minimal extent. Indeed, to ensure the best possible investigatory results with the least amount of variance between centers, the Asthma Clinical Research Network (ACRN) instituted strict written criteria that were continually overviewed. This article describes the ACRN's quality control of: clinic procedures and equipment, training and certification of study personnel, site visit evaluations, and the procedures of bronchoscopy and sputum induction. The examples given in this report demonstrate how various aspects of our investigation are performed.

Relationship between airway inflammation, hyperresponsiveness, and obstruction in asthma.

BACKGROUND: Although the role of eosinophils in airway inflammation in chronic asthma has been extensively studied, a role for neutrophils has not been well characterized. Furthermore, prior studies have not systematically sought or controlled for factors that might confound the relationship between cellular markers of inflammation and physiologic measures of airway function. OBJECTIVE: The purpose of this study was to determine whether eosinophilic and neutrophilic inflammation independently contribute to abnormalities of airway function in asthma. METHODS: Multivariate analysis of data collected during screening and enrollment of 205 asthmatic adults for clinical trials was conducted to examine the relationships between cellular inflammation in induced sputum and FEV(1) and methacholine responsiveness (PC(20)) while confounding factors were controlled for. RESULTS: We found that age, sex, ethnicity, and use of inhaled corticosteroids were important confounding factors of the relationship between cellular inflammation and airway function. When these factors were controlled for, multivariate analysis showed that eosinophil percentage in induced sputum is independently associated with lower FEV(1) and lower PC(20) (P = .005 and P = .005, respectively). In the same models, increased sputum neutrophil percentage is independently associated with lower FEV(1) (P = .038) but not with PC(20) (P = .49). CONCLUSIONS: These results suggest that both eosinophilic inflammation and neutrophilic inflammation independently contribute to abnormalities of FEV(1) in asthma. Therapies directed specifically at control of neutrophilic inflammation might be useful in improving airway caliber in patients with chronic asthma.

Allergen challenge causes inflammation but not goblet cell degranulation in asthmatic subjects.

BACKGROUND: An allergen challenge to the airways of sensitized mice causes eosinophilic airway inflammation and degranulation of goblet cells, which lead to airway obstruction. However, whether allergen challenge causes a similar pattern of airway inflammation and goblet cell degranulation in human beings is unknown. OBJECTIVE: The purpose of this study was to determine whether allergen challenge increases airway inflammatory cells and causes goblet cell degranulation in human subjects with asthma. METHODS: In bronchial biopsy specimens taken from 8 asthmatic subjects at 1 and 24 hours after allergen challenge, we measured eosinophil and neutrophil numbers as indicators of inflammation. We also measured goblet cell mucin stores and the amounts of secreted mucin in bronchial lavage as indicators of goblet cell degranulation. RESULTS: Airway eosinophil numbers at both 1 and 24 hours after allergen challenge were twice as high as those after diluent challenge. Changes in neutrophil numbers were smaller and statistically insignificant. Goblet cell mucin stores measured in tissue stained with alcian blue/periodic acid-Schiff did not decrease significantly from baseline to 1 hour and actually tended to increase at 24 hours. This increase was significant in the subgroup of subjects with normal stored mucin levels at baseline. Mucin-like glycoprotein concentrations in bronchial lavage did not change significantly at either time point. CONCLUSION: Although allergen challenge in asthmatic subjects increases airway eosinophil numbers as early as 1 hour after challenge, this inflammatory response does not cause goblet cell degranulation. In fact, in subjects with normal baseline mucin stores, allergen challenge increases goblet cell mucin stores.

A novel method of gene transcript profiling in airway biopsy homogenates reveals increased expression of a Na+-K+-Cl- cotransporter (NKCC1) in asthmatic subjects.

Comprehensive and systematic analysis of airway gene expression represents a strategy for addressing the multiple, complex, and largely untested hypotheses that exist for disease mechanisms, including asthma. Here, we report a novel real-time PCR-based method specifically designed for quantification of multiple low-abundance transcripts using as little as 2.5 fg of total RNA per gene. This method of gene expression profiling has the same specificity and sensitivity as RT-PCR and a throughput level comparable to low-density DNA microarray hybridization. In this two-step method, multiplex RT-PCR is successfully combined with individual gene quantification via real-time PCR on generated cDNA product. Using this method, we measured the expression of 75 genes in bronchial biopsies from asthmatic versus healthy subjects and found expected increases in expression levels of Th2 cytokines and their receptors in asthma. Surprisingly, we also found increased gene expression of NKCC1--a Na+-K+-Cl- cotransporter. Using immunohistochemical method, we confirmed increased protein expression for NKCC1 in the asthmatic subject with restricted localization to goblet cells. These data validate the new transcriptional profiling method and implicate NKCC1 in the pathophysiology of mucus hypersecretion in asthma. Potential applications for this method include transcriptional profiling in limited numbers of laser captured cells and validation of DNA microarray data in clinical specimens.

Modifications in the "upper" velbenamine part of the Vinca alkaloids have major implications for tubulin interacting activities.

Vinca alkaloids represent a chemical class of major interest in cancer chemotherapy. The lead compounds vinblastine and vincristine have been employed in clinical practice for more than thirty years and remain widely used to this day. Several hundred derivatives have been synthesised and evaluated for their pharmacological activities, the majority being modified in the vindoline moiety, bearing several reactive centers. These efforts led to the identification of the amido derivative vindesine, registered in Europe in 1980 and now available in several countries. Then novel chemistry permitted the semisynthesis of derivatives modified in the velbenamine "upper" part of the molecule, creating a new potential in the Vinca alkaloids medicinal chemistry: as a result, vinorelbine, obtained by C' ring contraction of anhydrovinblastine, and is now marketed worldwide. Several strategies aimed at the total synthesis of vinblastine derivatives have been investigated, giving the opportunity to design rationally certain compounds. Modifications in the D' ring appeared to induce dramatic changes in the tubulin interactions. These observations have been confirmed recently by the identification of unprecedented pharmacological properties exerted by the novel fluorinated Vinca alkaloid, vinflunine. This review will focus more specifically on derivatives which have been modified in the velbenamine part, with the aim of inducing different chemical and pharmacological properties.