Perinatal inflammation is associated with respiratory morbidity. Immune modulation of brainstem respiratory control centers may provide a link for this pathobiology. We exposed 11-day old rats to intratracheal lipopolysaccharide (LPS, 0.5 µg/g) to test the hypothesis that intrapulmonary inflammation increases expression of the proinflammatory cytokine IL-1β within respiratory-related brainstem regions. Intratracheal LPS resulted in a 32% increase in IL-1β protein expression in the medulla oblongata. In situ hybridization showed increased intensity of IL-1β mRNA but no change in neuronal numbers. Co-localization experiments showed that hypoglossal neurons express IL-1β mRNA and immunostaining showed a 43% increase in IL-1β protein-expressing cells after LPS exposure. LPS treatment also significantly increased microglial cell numbers though they did not express IL-1β mRNA. LPS-induced brainstem expression of neuronal IL-1β mRNA and protein may have implications for our understanding of the vulnerability of neonatal respiratory control in response to a peripheral pro-inflammatory stimulus.
Brainstem; neuro-inflammation; in situ hybridization; lipopolysaccharide; respiratory control
Nematode anthelminthic resistance is widespread for the 3 major drug classes commonly used in agriculture: benzamidazoles, macrocyclic lactones, and nicotinic agonists e.g. levamisole. In parasitic nematodes the genetics of resistance are unknown other than to the benzimidazoles which primarily involve a single gene. In previous work with a levamisole resistant Oesophagostomum dentatum isolate, the nicotinic acetylcholine receptor (nAChR) exhibited decreased levamisole sensitivity. Here, using a transcriptomic approach on the same isolate, we investigate whether that decreased nAChR sensitivity is achieved via a 1-gene mechanism involving 1 of 27 nAChR pathway genes. 3 nAChR receptor subunit genes exhibited ≥ 2-fold change in transcript abundance: acr-21 and acr-25 increased, and unc-63 decreased. 4 SNPs having a ≥ 2-fold change in frequency were also identified. These data suggest that resistance is likely polygenic, involving modulated abundance of multiple subunits comprising the heteropentameric nAChR, and is not due to a simple 1-gene mechanism.
resistance; levamisole; nAChR; nodular worm
Lung diseases, such as bronchopulmonary dysplasia (BPD), wheezing, and asthma, remain significant causes of morbidity and mortality in the pediatric population, particularly in the setting of premature birth. Pulmonary outcomes in these infants are highly influenced by perinatal exposures including prenatal inflammation, postnatal intensive care unit interventions, and environmental agents. Here, there is strong evidence that perinatal supplemental oxygen administration has significant effects on pulmonary development and health. This is of particular importance in the preterm lung, where premature exposure to room air represents a hyperoxic insult that may cause harm to a lung primed to develop in a hypoxic environment. Preterm infants are also subject to increased episodes of hypoxia, which may also result in pulmonary damage and disease. Here, we summarize current understanding of the effects of oxygen on the developing lung and how low vs. high oxygen may predispose to pulmonary disease that may extend even into adulthood. Better understanding of the underlying mechanisms will help lead to improved care and outcomes in this vulnerable population.
Neonatal; Bronchopulmonary Dysplasia; Asthma; Hypoxia; Hyperoxia
Despite changes in the epidemiology of bronchopulmonary dysplasia [BPD], longer term morbidity, particularly in the form of airway dysfunction, remains a substantial problem in former preterm infants. The stage for this respiratory morbidity may begin as early as the transition from fetal to neonatal life. Newer therapeutic approaches for BPD should be directed toward minimizing this longer term respiratory morbidity. Neonatal animal models focused primarily on hyperoxic exposure may provide important insights into the pathogenesis of longer term airway hyperreactivity in this population.
bronchopulmonary dysplasia; neonatal airway function; pediatric asthma
•Special Issue from the “Anthelmintics: From Discovery to Resistance” meeting, San Francisco, February 2014.•Meeting themes: drug discovery, modes of action and resistance.•Human and veterinary parasites covered.•Academic and industrial attendees.
The scientific meeting entitled ‘Anthelmintics: From Discovery to Resistance’ was held in San Francisco in February 2014. The themes of the meeting were drug discovery, modes of action and resistance. Both human and veterinary parasites were covered in the oral and poster presentations. The attendees were from both academic and industrial backgrounds. In the present article we introduce a number of the papers that emerged from the meeting. Several of the papers covered current drug discovery efforts underway worldwide, with some specific examples focusing on ion channels, protein kinases and cysteine proteases. These efforts included the repurposing of known drugs as well as the discovery of novel actives. Two papers described recently-developed whole-organism screening techniques. Finally, we introduce several papers looking at mechanisms and management of drug resistance in human and veterinary parasites.
Anthelmintics; Resistance; Drug discovery; Scientific meeting
Diethylcarbamazine is a drug that is used for the treatment of filariasis in humans and animals; it also has effects on intestinal nematodes, but its mechanism of action remains unclear. Emodepside is a resistance-busting anthelmintic approved for treating intestinal parasitic nematodes in animals. The novel mode of action and resistance-breaking properties of emodepside has led to its use against intestinal nematodes of animals, and as a candidate drug for treating filarial parasites. We have previously demonstrated effects of emodepside on SLO-1 K+-like currents in Ascaris suum. Here, we demonstrate that diethylcarbamazine, which has been proposed to work through host mediated effects, has direct effects on a nematode parasite, Ascaris suum. It increases activation of SLO-1 K+ currents and potentiates effects of emodepside. Our results suggest consideration of the combination of emodepside and diethylcarbamazine for therapy, which is predicted to be synergistic. The mode of action of diethylcarbamazine may involve effects on parasite signaling pathways (including nitric oxide) as well as effects mediated by host inflammatory mediators.
Filarial parasites and soil-transmitted nematodes (STNs) are Neglected Tropical Diseases (NTDs) that affect millions of people in the developing world. There is an urgent need for novel drugs and improved use of existing drugs, because of concerns about the development of resistance. The mode of action of one of these drugs, diethylcarbamazine, remains unclear, despite the fact that it has been used for a long time for treatment and prevention of filariae and STNs. The resistance-busting anthelmintic emodepside also has effects against filariae and STNs, with a mode of action that involves activation of nematode SLO-1 K+ channels. The effects of both diethylcarbamazine and emodepside may be increased by inflammatory mediators, which suggests that the effects of diethylcarbamazine and emodepside will be additive. We used our Ascaris suum preparation to test the activation of SLO-1 K+ channels by diethylcarbamazine and its potentiating effect on emodepside. Our results suggest potential for diethylcarbamazine and emodepside in combination therapy for parasitic nematodes.
Rationale: The role of airway microbiome in corticosteroid response in asthma is unknown.
Objectives: To examine airway microbiome composition in patients with corticosteroid-resistant (CR) asthma and compare it with patients with corticosteroid-sensitive (CS) asthma and normal control subjects and explore whether bacteria in the airways of subjects with asthma may direct alterations in cellular responses to corticosteroids.
Methods: 16S rRNA gene sequencing was performed on bronchoalveolar lavage (BAL) samples of 39 subjects with asthma and 12 healthy control subjects. In subjects with asthma, corticosteroid responsiveness was characterized, BAL macrophages were stimulated with pathogenic versus commensal microorganisms, and analyzed by real-time polymerase chain reaction for the expression of corticosteroid-regulated genes and cellular p38 mitogen-activated protein kinase (MAPK) activation.
Measurements and Main Results: Of the 39 subjects with asthma, 29 were CR and 10 were CS. BAL microbiome from subjects with CR and CS asthma did not differ in richness, evenness, diversity, and community composition at the phylum level, but did differ at the genus level, with distinct genus expansions in 14 subjects with CR asthma. Preincubation of asthmatic airway macrophages with Haemophilus parainfluenzae, a uniquely expanded potential pathogen found only in CR asthma airways, resulted in p38 MAPK activation, increased IL-8 (P < 0.01), mitogen-activated kinase phosphatase 1 mRNA (P < 0.01) expression, and inhibition of corticosteroid responses (P < 0.05). This was not observed after exposure to commensal bacterium Prevotella melaninogenica. Inhibition of transforming growth factor-β–associated kinase-1 (TAK1), upstream activator of MAPK, but not p38 MAPK restored cellular sensitivity to corticosteroids.
Conclusions: A subset of subjects with CR asthma demonstrates airway expansion of specific gram-negative bacteria, which trigger TAK1/MAPK activation and induce corticosteroid resistance. TAK1 inhibition restored cellular sensitivity to corticosteroids.
microbiome; asthma; corticosteroids
In asthma and other diseases, vitamin D insufficiency is associated with adverse outcomes. It is not known if supplementing inhaled corticosteroids with oral vitamin D3 improves outcomes in patients with asthma and vitamin D insufficiency.
To evaluate if vitamin D supplementation would improve the clinical efficacy of inhaled corticosteroids in patients with symptomatic asthma and lower vitamin D levels.
DESIGN, SETTING, AND PARTICIPANTS
The VIDA (Vitamin D Add-on Therapy Enhances Corticosteroid Responsiveness in Asthma) randomized, double-blind, parallel, placebo-controlled trial studying adult patients with symptomatic asthma and a serum 25-hydroxyvitamin D level of less than 30 ng/mL was conducted across 9 academic US medical centers in the National Heart, Lung, and Blood Institute’s AsthmaNet network, with enrollment starting in April 2011 and follow-up complete by January 2014. After a run-in period that included treatment with an inhaled corticosteroid, 408 patients were randomized.
Oral vitamin D3 (100 000 IU once, then 4000 IU/d for 28 weeks; n = 201) or placebo (n = 207) was added to inhaled ciclesonide (320 µg/d). If asthma control was achieved after 12 weeks, ciclesonide was tapered to 160 µg/d for 8 weeks, then to 80 µg/d for 8 weeks if asthma control was maintained.
MAIN OUTCOMES AND MEASURES
The primary outcome was time to first asthma treatment failure (a composite outcome of decline in lung function and increases in use of β-agonists, systemic corticosteroids, and health care).
Treatment with vitamin D3 did not alter the rate of first treatment failure during 28 weeks (28%[95% CI, 21%-34%] with vitamin D3 vs 29% [95% CI, 23%–35%] with placebo; adjusted hazard ratio, 0.9 [95% CI, 0.6–1.3]). Of 14 prespecified secondary outcomes, 9 were analyzed, including asthma exacerbation; of those 9, the only statistically significant outcome was a small difference in the overall dose of ciclesonide required to maintain asthma control (111.3 µg/d [95% CI, 102.2–120.4 µg/d] in the vitamin D3 group vs 126.2 µg/d [95% CI, 117.2–135.3 µg/d] in the placebo group; difference of 14.9 µg/d [95% CI, 2.1–27.7 µg/d]).
CONCLUSIONS AND RELEVANCE
Vitamin D3 did not reduce the rate of first treatment failure or exacerbation in adults with persistent asthma and vitamin D insufficiency. These findings do not support a strategy of therapeutic vitamin D3 supplementation in patients with symptomatic asthma.
clinicaltrials.gov Identifier: NCT01248065
Tiotropium has activity as an asthma controller. However, predictors of a positive response to tiotropium have not been described.
To describe individual and differential response of patients with asthma to salmeterol and tiotropium, when added to an ICS, as well as predictors of a positive clinical response.
Data from the double-blind, three-way crossover NHLBI Asthma Clinical Research Network’s TALC trial (ClinicalTrials.gov number, NCT00565266) were analyzed for individual and differential treatment responses to salmeterol and tiotropium, and predictors of a positive response to the endpoints FEV1, morning peak expiratory flow (AM PEF), and asthma control days (ACDs).
While approximately equal numbers of patients showed a differential response to salmeterol and tiotropium in terms of AM PEF (90 and 78, respectively), and ACDs (49 and 53, respectively), more showed a differential response to tiotropium for FEV1 (104) than salmeterol (62). An acute response to a short-acting bronchodilator, especially albuterol, predicted a positive clinical response to tiotropium for FEV1 (OR 4.08 [CI 2.00–8.31], P < 0.001) and AM PEF (OR 2.12 [CI 1.12–4.01], P = 0.021), as did a decreased FEV1/FVC ratio (FEV1 response increased 0.39% of baseline for every 1% decrease in the FEV1/FVC ratio). Higher cholinergic tone was also a predictor, while ethnicity, gender, atopy, IgE Level, sputum eosinophils, FENO, asthma duration, and BMI were not.
While these results need confirmation, predictors of a positive clinical response to tiotropium include a positive response to albuterol and airway obstruction, factors which could help identify appropriate patients for this therapy.
asthma; tiotropium; salmeterol; responder analysis; predictor of response
Small airway changes and dysfunction contribute importantly to airway obstruction in chronic obstructive pulmonary disease (COPD), which is currently treated with inhaled corticosteroids (ICS) and long-acting bronchodilators at Global initiative for Obstructive Lung Disease (GOLD) grades 2–4. This retrospective matched cohort analysis compared effectiveness of a representative small-particle ICS (extrafine beclomethasone) and larger-particle ICS (fluticasone) in primary care patients with COPD.
Patients and methods
Smokers and ex-smokers with COPD ≥40 years old initiating or stepping-up their dose of extrafine beclomethasone or fluticasone were matched 1:1 for demographic characteristics, index prescription year, concomitant therapies, and disease severity during 1 baseline year. During 2 subsequent years, we evaluated treatment change and COPD exacerbations, defined as emergency care/hospitalization for COPD, acute oral corticosteroids, or antibiotics for lower respiratory tract infection.
Mean patient age was 67 years, 57%–60% being male. For both initiation (n=334:334) and step-up (n=189:189) patients, exacerbation rates were comparable between extrafine beclomethasone and fluticasone cohorts during the 2 year outcome period. Odds of treatment stability (no exacerbation or treatment change) were significantly greater for patients initiating extrafine beclomethasone compared with fluticasone (adjusted odds ratio 2.50; 95% confidence interval, 1.32–4.73). Median ICS dose exposure during 2 outcome years was significantly lower (P<0.001) for extrafine beclomethasone than fluticasone cohorts (315 μg/day versus 436 μg/day for initiation, 438 μg/day versus 534 μg/day for step-up patients).
We observed that small-particle ICS at significantly lower doses had comparable effects on exacerbation rates as larger-particle ICS at higher doses, whereas initiation of small-particle ICS was associated with better odds of treatment stability during 2-years’ follow-up.
COPD exacerbation; extrafine particle; matched cohort analysis; real life; small airways
Wheezing and asthma are significant clinical problems for infants and young children, particularly following premature birth. Recurrent wheezing in infants can progress to persistent asthma. As in adults, altered airway structure (remodeling) and function (increased bronchoconstriction) are also important in neonatal and pediatric airway diseases. Accumulating evidence suggests that airway disease in children is influenced by perinatal factors including perturbations in normal fetal lung development, postnatal interventions in the intensive care unit, and environmental and other insults in the neonatal period. Here, in addition to genetics, maternal health, environmental processes, innate immunity, and impaired lung development/function can all influence pathogenesis of airway disease in children. We summarize current understanding of how prenatal and postnatal factors can contribute to development of airway diseases in neonates and children. Understanding these mechanisms will help identify and develop novel therapies for childhood airway diseases.
Infant; Child; Perinatal; Asthma; Wheezing; Remodeling; Bronchoconstriction
Recurrent episodes of hypoxemia may affect the growth, cardiac function, neurologic outcome, and survival of infants with bronchopulmonary dysplasia (BPD). As oral feeding might stress these infants by compromising pulmonary function even after hospital discharge, we measured oxygen saturation (Sao2) via pulse oximetry before, during the initial 10 minutes of, and immediately after oral feeding in 11 patients with BPD, 12 very low birth weight infants, and 23 healthy full-term infants. All infants with BPD had been previously discharged from the hospital after weaning from supplemental oxygen. Studies were done at a mean postconceptional age of 43 weeks while the infants were fed at home by one of their parents. Levels of Sao2 for the three groups were comparable before and during feeds. After feeding, the infants with BPD had significantly lower mean levels of Sao2 (84 ± 8% [SD] vs 93 ± 4% and 93 ± 3%, respectively; P < .01). They also spent more time after feeding with an Sao2 <90% (64 ± 34% of time vs 27 ± 33% for the very low birth weight and 22 ± 20% for the term group; P < .01) and greater time with an Sao2 <80% (37 ± 28% vs 4 ± 10% and 4 ± 8%, respectively; P < .01). Desaturation in infants with BPD was related to larger volume and faster oral intake during feeding. Thus, the data indicate that desaturation after feeding remains a recurrent problem for survivors of BPD after discharge. Individual approaches which incorporate parental education and behavioral interventions might decrease the risk of significant hypoxemia during oral feeding in infants with BPD.
Perinatal sepsis and inflammation trigger lung and brain injury in preterm infants, and associated apnea of prematurity. We hypothesized that endotoxin exposure in the immature lung would upregulate proinflammatory cytokine mRNA expression in the medulla oblongata and be associated with impaired respiratory control. Lipopolysaccharide (LPS, 0.1 mg/kg) or saline was administered intratracheally to rat pups and medulla oblongatas were harvested for quantifying expression of mRNA for proinflammatory cytokines. LPS-exposure significantly increased medullary mRNA for IL-1β and IL-6, and vagotomy blunted this increase in IL-1β, but not IL-6. Whole-body flow plethysmography revealed that LPS-exposed pups had an attenuated ventilatory response to hypoxia both before and after carotid sinus nerve transection. Immunochemical expression of IL-1β within the nucleus of the solitary tract and area postrema was increased after LPS-exposure. In summary, intratracheal endotoxin-exposure in rat pups is associated with upregulation of proinflammatory cytokines in the medulla oblongata that is vagally-mediated for IL-1β and associated with an impaired hypoxic ventilatory response.
Intermittent hypoxic episodes are typically a consequence of immature respiratory control and remain a troublesome challenge for the neonatologist. Furthermore, their frequency and magnitude are underestimated by clinically employed pulse oximeter settings. In extremely low birth weight infants the incidence of intermittent hypoxia progressively increases over the first 4 weeks of postnatal life, with a subsequent plateau followed by a slow decline beginning at weeks 6–8. Such episodic hypoxia/reoxygenation has the potential to sustain a proinflammatory cascade with resultant multisystem morbidity. This morbidity includes retinopathy of prematurity and impaired growth, as well as possible longer-term cardiorespiratory instability and poor neurodevelopmental outcome. Therapeutic approaches for intermittent hypoxic episodes comprise determination of optimal baseline saturation and careful titration of supplemental inspired oxygen, as well as xanthine therapy to prevent apnea of prematurity. In conclusion, characterization of the pathophysiologic basis for such intermittent hypoxic episodes and their consequences during early life is necessary to provide an evidence-based approach to their management.
Intermittent hypoxic episodes; Pulse oximetry; Extremely low birth weight infants
Perinatal inflammation and neonatal sepsis trigger lung and brain injury. We hypothesized that endotoxin exposure in the immature lung upregulates proinflammatory cytokine expression in the brainstem and impairs respiratory control. Lipopolysaccharide (LPS) or saline was administered intratracheally to vagal intact or denervated rat pups. LPS increased brainstem IL-1β and vagotomy blunted this response. There was an attenuated ventilatory response to hypoxia and increased brainstem IL-1β expression after LPS.
Intratracheal endotoxin exposure in rat pups is associated with upregulation of IL-1β in the brainstem that is vagally mediated and associated with an impaired hypoxic ventilatory response.
Brainstem cytokines; Hypoxic ventilatory response; Neonatal respiratory control
•We report on the Consortium for Anthelmintic Resistance and Susceptibility 2013 meeting.•Recent advances in the identification of markers for anthelmintic resistance are described.•The use of markers for benzimidazole resistance in field studies with veterinary and human nematodes.•The application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.
Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthelmintic Resistance and Susceptibility (CARS) provides a forum for scientists to meet and discuss the latest developments in the search for molecular markers of anthelmintic resistance. Such markers are important for detecting drug resistant worm populations, and indicating the likely impact of the resistance on drug efficacy. The molecular basis of resistance is also important for understanding how anthelmintics work, and how drug resistant populations arise. Changes to target receptors, drug efflux and other biological processes can be involved. This paper reports on the CARS group meeting held in August 2013 in Perth, Australia. The latest knowledge on the development of molecular markers for resistance to each of the principal classes of anthelmintics is reviewed. The molecular basis of resistance is best understood for the benzimidazole group of compounds, and we examine recent work to translate this knowledge into useful diagnostics for field use. We examine recent candidate-gene and whole-genome approaches to understanding anthelmintic resistance and identify markers. We also look at drug transporters in terms of providing both useful markers for resistance, as well as opportunities to overcome resistance through the targeting of the transporters themselves with inhibitors. Finally, we describe the tools available for the application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.
Anthelmintic resistance; Anthelmintic drugs; Molecular markers; Anthelmintic targets; Receptors
Strategies employed by parasites to establish infections are poorly understood. The host-parasite interface is maintained through a molecular dialog that, among other roles, protects parasites from host immune responses. Parasite excretory/secretory products (ESP) play major roles in this process. Understanding the biology of protein secretion by parasites and their associated functional processes will enhance our understanding of the roles of ESP in host-parasite interactions.
ESP was collected after culturing 10 adult female Ascaris suum. Perienteric fluid (PE) and uterine fluid (UF) were collected directly from adult females by dissection. Using SDS-PAGE coupled with LC-MS/MS, we identified 175, 308 and 274 proteins in ESP, PE and UF, respectively. Although many proteins were shared among the samples, the protein composition of ESP was distinct from PE and UF, whereas PE and UF were highly similar. The distribution of gene ontology (GO) terms for proteins in ESP, PE and UF supports this claim. Comparison of ESP composition in A. suum, Brugia malayi and Heligmosoides polygyrus showed that proteins found in UF were also secreted by males and by larval stages of other species, suggesting that multiple routes of secretion may be used for homologous proteins. ESP composition of nematodes is both phylogeny- and niche-dependent.
Analysis of the protein composition of A. suum ESP and UF leads to the conclusion that the excretory-secretory apparatus and uterus are separate routes for protein release. Proteins detected in ESP have distinct patterns of biological functions compared to those in UF. PE is likely to serve as the source of the majority of proteins in UF. This analysis expands our knowledge of the biology of protein secretion from nematodes and will inform new studies on the function of secreted proteins in the orchestration of host-parasite interactions.
Ascaris lumbricoides, the most prevalent metazoan parasite of humans, is a public health concern in resource-limited countries. Survival of this parasite in its host is mediated at least in part by parasite materials secreted into the host. Little is known about the composition of these secretions; defining their contents and functions will illuminate host-parasite interactions that lead to parasite establishment. Ascaris suum, a parasite of pigs, was used as a model organism because its genome has been sequenced and it is very closely related to A. lumbricoides. Excretory/secretory products (ESP), uterine fluid (UF) and perienteric fluid (PE) were collected from adult A. suum. Proteins were subjected to LC-MS/MS. ESP proteins (the ‘secretome’) included many also present in UF. Proteins in ESP but not in UF had considerably different characteristics than those in PE or UF, which were similar to each other. We conclude that proteins released from the secretory apparatus have distinct patterns of biological function and that UF proteins are likely derived from PE. Comparing the protein composition of A. suum ESP to ESP from B. malayi and H. polygyrus suggests that the secretome is conserved at the level of both phylogeny and host predilection site.
The role of CD8 T lymphocytes in the pathogenesis of asthma is not well understood. We investigated whether a subset of IL-13-producing BLT1-positive CD8 T lymphocytes is present in asthmatic airways and is associated with impaired lung function.
Bronchoalveolar lavage (BAL) cells were obtained from asthmatic (n=39) and healthy control (n=28) subjects. Cells were stimulated with phorbol ester and ionomycin in the presence of brefeldin A and stained for CD8, BLT1 and intracellular IL-13. The frequency of IL-13-producing BLT1-positive CD8 T lymphocytes was compared between the two groups and related to lung function, serum IgE levels and reticular basement membrane (RBM) thickness.
A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 was detected in the airways of all asthmatic subjects. The frequency of this subset among recovered lymphocytes was significantly higher in the airways of asthmatic subjects compared to controls (mean ± SEM: 16.2 ± 1.4 vs. 5.3 ± 0.5, respectively, p < 0.001), and correlated positively with serum IgE levels and RBM thickness. More importantly, the frequency of CD8 T lymphocytes co-expressing BLT1 and IL-13 was inversely related to FEV1 and FEF[25-75] percent predicted values (p<0.001).
A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 is present in the airways of asthmatics. The accumulation of these cells is associated with airway obstruction, suggesting that they may play a significant pathogenic role in bronchial asthma.
Asthma; CD8 lymphocytes; immunoglobulin E; interleukin-13; leukotriene B4 receptor
Infection with Mycoplasma pneumoniae in asthma can occur both acutely and chronically with an associated Th2 inflammatory response and/or increased numbers of bronchial mast cells. Mast cells have previously been shown to promote mycoplasma clearance in mice; however, it is unknown whether mast cells would aid M. pneumoniae clearance under allergic conditions.
Our aim was to determine the impact of allergic inflammation on mast cell-mediated lung M. pneumoniae clearance. Furthermore, as we have previously demonstrated an essential role for IL-6 in lung M. pneumoniae clearance we also investigated the role of mast cell-derived IL-6.
Mast cell deficient WBB6F1/J-KitW/KitW-v mice were challenged with ovalbumin to induce airway inflammation prior to M. pneumoniae infection. The role of mast cell-derived IL-6 in bacterial clearance was further investigated by reconstitution of mast cell deficient mice with IL-6-/- mast cells.
Allergic mast cell deficient mice exhibited increased lung M. pneumoniae burden compared to control littermates. Intravenous adoptive transfer of wild type and IL-6-/- mast cells significantly improved M. pneumoniae clearance in mast cell deficient mice. Acutely after M. pneumoniae infection, allergen-challenged mast cell deficient mice had increased levels of the pro-inflammatory cytokines IL-6 and TNF-α in the BAL fluid. The total number of neutrophils was also increased in mast cell deficient mice.
Our results establish that mast cells aid host defense against M. pneumoniae in an allergic setting and that while IL-6 is necessary for lung M. pneumoniae clearance, mast cell-derived IL-6 is not required.
Asthma; host defense; innate immunity; mast cells; Mycoplasma pneumoniae
StartectR is a novel anthelmintic combination of derquantel and abamectin. It is hypothesized that derquantel and abamectin interact pharmacologically. We investigated the effects of derquantel, abamectin and their combination on somatic muscle nicotinic acetylcholine receptors and pharyngeal muscle glutamate gated chloride receptor channels of Ascaris suum. We used muscle-strips to test the effects of abamectin, derquantel, and abamectin + derquantel together on the contraction responses to different concentrations of acetylcholine. We found that abamectin reduced the response to acetylcholine, as did derquantel. In combination (abamectin + derquantel), inhibition of the higher acetylcholine concentration responses was statistically greater than the predicted additive effect. A two-micropipette current-clamp technique was used to study electrophysiological effects of the anthelmintics on: 1) acetylcholine responses in somatic muscle and; 2) on L-glutamate responses in pharyngeal preparations. On somatic muscle, derquantel (0.1 - 30 μM) produced a potent (IC50 0.22, CI 0.18-0.28 μM) reversible antagonism of acetylcholine depolarizations. Abamectin (0.3 μM) produced a slow onset inhibition of acetylcholine depolarizations. We compared effects of abamectin and derquantel on muscle preparations pretreated for 30 minutes with these drugs. The effect of the combination was significantly greater than the predicted additive effect of both drugs at higher acetylcholine concentrations. On the pharynx, application of derquantel produced no significant effect by itself or on responses to abamectin and L-glutamate. Abamectin increased the input conductance of the pharynx (EC50 0.42, CI 0.13-1.36 μM). Our study demonstrates that abamectin and derquantel interact at nicotinic acetylcholine receptors on the somatic muscle and suggested synergism can occur.
abamectin; derquantel; combination; interaction; nAChRs; GluCls
This paper reports on the development of a lens-less and image-sensor-less micro-electro-fluidic (MEF) approach for real-time monitoring of the locomotion of microscopic nematodes. The technology showed promise for overcoming the constraint of the limited field of view of conventional optical microscopy, with a relatively low cost, good spatial resolution, and high portability. The core of the device was microelectrode grids formed by orthogonally arranging two identical arrays of microelectrode lines. The two microelectrode arrays were spaced by a microfluidic chamber containing a liquid medium of interest. As a nematode (e.g., Caenorhabditis elegans) moved inside the chamber, the invasion of its body parts into some intersection regions between the microelectrodes caused changes in electrical resistance of these intersection regions. The worm's presence at or absence from a detection unit was determined by a comparison between the measured resistance variation of this unit and a pre-defined threshold resistance variation. An electronic readout circuit was designed to address all detection units and read out their individual electrical resistance. By this means, it was possible to obtain the electrical resistance profile of the whole MEF grids, and thus, the physical pattern of the swimming nematode. We studied the influence of a worm's body on the resistance of an addressed unit. We also investigated how the full-frame scanning and readout rate of the electronic circuit and the dimensions of a detection unit posed an impact on the spatial resolution of the reconstructed images of the nematode. Other important issues, such as the manufacturing induced initial non-uniformity of the grids and the electrotaxic behaviour of nematodes, were also studied. A drug resistance screening experiment was conducted by using the grids with a good resolution of 30 × 30 μm2. The phenotypic differences in the locomotion behaviours (e.g., moving speed and oscillation frequency extracted from the reconstructed images with the help of software) between the wild-type (N2) and mutant (lev-8) C. elegans worms in response to different doses of the anthelmintic drug, levamisole. The locomotive parameters obtained by the MEF grids agreed well with those obtained by optical microscopy. Therefore, this technology will benefit the whole-animal assays by providing a structurally simple, potentially cost-effective device capable of tracking the movement and phenotypes of important nematodes in various microenvironments.
The cause of corticosteroid resistant asthma is unknown.
To perform gene microarray analyses using BAL cells from well-characterized corticosteroid resistant (CR) and sensitive (CS) asthmatics to elucidate the differential expression of genes that contribute to the development of corticosteroid resistance.
The patients were characterized as CR or CS based on FEV1% predicted improvement after one week course of oral prednisone. Expression of selected gene targets was verified by real time PCR and by ELISA.
Microarray analyses demonstrated significantly higher levels (over three-fold increase, p<0.05) of transcripts for TNFα, IL-1α, IL-1β, IL-6, CXCL1, CXCL2, CXCL3, CXCL8 (IL-8), CCL3, CCL4, CCL20 in BAL cells of CR asthmatics. These findings, confirmed by RT-PCR in additional BAL samples, were consistent with classical macrophage activation by bacterial products. In contrast, markers of alternatively-activated macrophages, Arginase I and CCL24, were decreased. Genes associated with activation of the LPS signaling pathway (EGR1, DUSP2, MAIL, TNFAIP3) were significantly elevated in CR BAL samples (p<0.05). These patients had significantly higher amounts (1444.0±457.3 pg per mg of total protein) of LPS in BAL fluid than CS asthmatics (270.5±216.0 pg; p<0.05) as detected by LAL assay and confirmed by gas chromatography mass spectrometry analysis. Pronged exposure to LPS induced functional steroid resistance to dexamethasone (DEX) in normal monocytes, demonstrated by persistently elevated IL-6 levels in the presence of DEX.
Classical macrophage activation and induction of LPS signaling pathways along with high endotoxin levels detected in BAL fluid from CR asthmatics suggest that LPS exposure may contribute to CR asthma.
corticosteroids; asthma; resistance; genes; endotoxin
Preterm infants are subject to fluctuations in blood gas status associated with immature respiratory control. Intermittent hypoxia during early postnatal life has been shown to increase chemoreceptor sensitivity and destabilize the breathing pattern; however, intermittent hypercapnia remains poorly studied. Therefore, to test the hypothesis that intermittent hypercapnia results in altered respiratory control, we examined the effects of daily exposure to intermittent hypercapnia on the ventilatory response to subsequent hypercapnic and hypoxic exposure in neonatal rat pups. Exposure cycles consisted of 5 min of intermittent hypercapnia (5% CO2, 21% O2, balance N2) followed by 10 min of normoxia. Rat pups were exposed to 18 exposure cycles each day for 1 week, from postnatal day 7 to 14. We analyzed diaphragm electromyograms (EMGs) from pups exposed to subsequent acute hypercapnic (5% CO2) and hypoxic (12% O2) challenges. In response to a subsequent hypercapnia challenge, there was no significant difference in the ventilatory response between control and intermittent hypercapnia-exposed groups. In contrast, intermittent hypercapnia-exposed rat pups showed an enhanced ventilatory response to hypoxic challenge with an increase in minute EMG to 118 ± 14% of baseline versus 107 ± 13% for control pups (p < 0.05). We speculate that prior hypercapnic exposure may increase peripheral chemoreceptor response to subsequent hypoxic exposures and result in perturbed neonatal respiratory control.
Hypoxia; Hypercapnia; Neonatal apnea; Chemoreceptors
One of the most abundant antioxidants in the lung is glutathione (GSH), a low molecular weight thiol, which functions to attenuate both oxidative stress and inflammation. GSH is concentrated in the epithelial lining fluid (ELF) of the lung and can be elevated in response to the increased oxidant burden from cigarette smoke (CS). However, the transporter(s) responsible for the increase in ELF GSH with cigarette smoke are not known. Three candidate apical GSH transporters in the lung are CFTR, BCRP and MRP2, but their potential role in ELF GSH transport in response to CS has not been investigated. In vitro, the inhibition of CFTR, BCRP, or MRP2 resulted in decreased GSH efflux in response to cigarette smoke extract. In vivo, mice deficient in CFTR, BCRP, or MRP2 were exposed to either air or acute CS. CFTR deficient mice had reduced basal and CS induced GSH in the ELF, while BCRP or MRP2 deficiency had no effect on ELF GSH basal or CS exposed levels. Furthermore, BCRP and MRP2 deficiencies had little effect on lung tissue GSH. These data indicate that CFTR is predominantly involved in maintaining basal ELF GSH and increasing ELF GSH in response to CS.