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1.  Proteomic Analysis of Adult Ascaris suum Fluid Compartments and Secretory Products 
Background
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.
Methodology/Principal Findings
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.
Conclusions/Significance
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.
Author Summary
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.
doi:10.1371/journal.pntd.0002939
PMCID: PMC4046973  PMID: 24901219
2.  IL-13-producing BLT1-positive CD8 cells are increased in asthma and are associated with airway obstruction 
Allergy  2013;68(5):666-673.
Background
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1111/all.12135
PMCID: PMC3630251  PMID: 23573812
Asthma; CD8 lymphocytes; immunoglobulin E; interleukin-13; leukotriene B4 receptor
3.  Why Do Former Preterm Infants Wheeze? 
The Journal of pediatrics  2012;162(3):10.1016/j.jpeds.2012.11.028.
doi:10.1016/j.jpeds.2012.11.028
PMCID: PMC3870137  PMID: 23260100
4.  Derquantel and Abamectin: Effects and interactions on isolated tissues of Ascaris suum 
Molecular and biochemical parasitology  2013;188(2):10.1016/j.molbiopara.2013.02.004.
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.
doi:10.1016/j.molbiopara.2013.02.004
PMCID: PMC3864555  PMID: 23523993
abamectin; derquantel; combination; interaction; nAChRs; GluCls
5.  Micro-electro-fluidic grids for nematodes: A lens-less, image-sensor-less approach for on-chip tracking of nematode locomotion 
Lab on a chip  2013;13(4):650-661.
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.
doi:10.1039/c2lc41174a
PMCID: PMC3587735  PMID: 23254956
6.  Corticosteroid Resistant Asthma is Associated with Classical Anti-Microbial Activation of Airway Macrophages 
Background
The cause of corticosteroid resistant asthma is unknown.
Objective
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/j.jaci.2008.07.007
PMCID: PMC3930345  PMID: 18774390
corticosteroids; asthma; resistance; genes; endotoxin
7.  CFTR is the primary known apical glutathione transporter involved in cigarette smoke induced adaptive responses in the lung 
Free radical biology & medicine  2012;52(7):1201-1206.
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.
doi:10.1016/j.freeradbiomed.2012.01.001
PMCID: PMC3920665  PMID: 22266045
8.  Markers for severity of illness associated with decreased snoring in toddlers born ELGA 
Aim
To describe the prevalence of paediatric sleep disordered breathing (SDB) symptoms in extremely low gestational age infants and identify neonatal risk factors, including early exposure to hypoxia and hyperoxia.
Methods
Patients <28 weeks gestation were monitored with high-resolution pulse oximetry. Hypoxia/hyperoxia variables were defined as percentage time of first 4 weeks of life that SaO2 < 80% or SaO2 > 98%, respectively. Parents completed part of the OSA-18 questionnaire for symptoms of SDB at 18–22 months. Logistic regression was used to test the association between risk factors and sleep symptoms.
Results
Of 182 patients recruited, 138 (76%) completed the questionnaire. The mean gestation was 26 weeks, and mean birth weight 887 grams. Loud snoring (21%) and restless sleep (24%) were the most prevalent symptoms. Female sex was associated with an increased risk of loud snoring (OR, 2.7; CI, 1.13–6.5). Prolonged mechanical ventilation, necrotizing enterocolitis and prolonged caffeine use, however, were inversely correlated with loud snoring. Neither neonatal hypoxia nor hyperoxia were associated with sleep symptoms.
Conclusions
While the prevalence of sleep disordered breathing symptoms is similar to reported rates, we found a sex difference not previously reported. Interestingly, markers for severity of illness show a pattern of being protective against loud snoring.
doi:10.1111/apa.12033
PMCID: PMC3650629  PMID: 23009601
Snoring; Sleep disordered breathing; Prematurity
9.  Mechanisms of Injury to the Preterm Lung and Airway: Implications for Long Term Pulmonary Outcome 
Neonatology  2012;101(4):345-352.
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.
doi:10.1159/000337355
PMCID: PMC3567481  PMID: 22940624
bronchopulmonary dysplasia; neonatal airway function; pediatric asthma
10.  An integrated fiber-optic microfluidic device for detection of muscular force generation of microscopic nematodes 
Lab on a chip  2012;12(18):3458-3466.
This paper reports development of an integrated fiber-optic microfluidic device for measuring muscular force of small nematode worms with high sensitivity, high data reliability, and simple device structure. A moving nematode worm squeezed through multiple detection points (DPs) created between a thinned single mode fiber (SMF) cantilever and a sine-wave channel with open troughs. The SMF cantilever was deflected by the normal force imposed by the worm, reducing optical coupling from the SMF to a receiving multimode fiber (MMF). Thus, multiple force data could be obtained for the worm-SMF contacts to verify to each other, improving data reliability. A noise equivalent displacement of the SMF cantilever was 0.28 μm and a noise equivalent force of the device was 143 nN. We demonstrated the workability of the device to detect muscular normal forces of the parasitic nematodes Oesophagotomum dentatum L3 larvae on the SMF cantilever. Also, we used this technique to measure force responses of levamisole-sensitive (SENS) and resistant (LERV) O. dentatum isolates in response to different doses of the anthelmintic drug, levamisole. The results showed that both of the isolates generated a larger muscular normal force when exposed to a higher concentration of levamisole. We also noticed muscular force phenotype differences between the SENS and LERV worms: the SENS muscles were more sensitive to levamisole than the LEVR muscles. The ability to quantify the muscular forces of small nematode worms will provide a new approach for screening mutants at single animal resolution. Also, the ability to resolve small differences in muscular forces in different environmental conditions will facilitate phenotyping different isolates of nematodes. Thus, the present technology can potentially benefit and advance the current whole animal assays.
doi:10.1039/c2lc40459a
PMCID: PMC3438457  PMID: 22824814
11.  Can Nitric Oxide Based Therapy Prevent Bronchopulmonary Dysplasia? 
Clinics in perinatology  2012;39(3):613-638.
doi:10.1016/j.clp.2012.06.011
PMCID: PMC3437658  PMID: 22954273
Inhaled Nitric Oxide (iNO); Bronchopulmonary Dysplasia (BPD); Neonate; Prematurity; S-nitrosylation; S-nitrosothiol (SNO); Ethyl Nitrite (ENO)
12.  Lung disease with anti-CCP antibodies but not rheumatoid arthritis or connective tissue disease 
Respiratory medicine  2012;106(7):1040-1047.
Summary
Objective
We sought to characterize a novel cohort of patients with lung disease, anti-cyclic citrullinated peptide (CCP) antibody positivity, without rheumatoid arthritis (RA) or other connective tissue disease (CTD).
Methods
The study sample included 74 subjects with respiratory symptoms, evaluated January 2008–January 2010 and found to have a positive anti-CCP antibody but no evidence for RA or other CTD. Each underwent serologic testing, pulmonary physiology testing, and thoracic high-resolution computed tomography (HRCT) scan as part of routine clinical evaluation.
Results
The majority of subjects were women, and most were former cigarette smokers. Four distinct radiographic phenotypes were identified: isolated airways disease (54%), isolated interstitial lung disease (ILD) (14%), mixed airways disease and ILD (26%), and combined pulmonary fibrosis with emphysema (7%). This cohort had a predominance of airways disease, either in isolation or along with a usual interstitial pneumonia-pattern of ILD. Among subjects with high-titer anti-CCP positivity (n=33), three developed the articular manifestations of RA during a median follow-up of 449 days.
Conclusion
We have described a unique cohort of patients with anti-CCP antibody positivity and lung disease in the absence of existing RA or other CTD. The lung phenotypic characteristics of this cohort resemble those of established RA and a few of these patients have developed articular RA within a short period of follow-up. The implications of a positive anti-CCP antibody among patients with lung disease but not RA are not yet known, but we believe requires further investigation.
doi:10.1016/j.rmed.2012.03.006
PMCID: PMC3753791  PMID: 22503074
Anti-cyclic citrullinated peptide; Rheumatoid arthritis; Interstitial lung disease; Lung diseases
13.  Vagal afferents modulate cytokine-mediated respiratory control at the neonatal medulla oblongata 
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.
doi:10.1016/j.resp.2011.03.003
PMCID: PMC3150618  PMID: 21397055
14.  Intermittent Hypoxic Episodes in Preterm Infants: Do They Matter? 
Neonatology  2011;100(3):303-310.
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.
doi:10.1159/000329922
PMCID: PMC3252018  PMID: 21986336
Intermittent hypoxic episodes; Pulse oximetry; Extremely low birth weight infants
16.  SPLUNC1 Deficiency Enhances Airway Eosinophilic Inflammation in Mice 
Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is enriched in normal airway lining fluid, but is significantly reduced in airway epithelium exposed to a Th2 cytokine milieu. The role of SPLUNC1 in modulating airway allergic inflammation (e.g., eosinophils) remains unknown. We used SPLUNC1 knockout (KO) and littermate wild-type (C57BL/6 background) mice and recombinant SPLUNC1 protein to determine the impact of SPLUNC1 on airway allergic/eosinophilic inflammation, and to investigate the underlying mechanisms. An acute ovalbumin (OVA) sensitization and challenge protocol was used to induce murine airway allergic inflammation (e.g., eosinophils, eotaxin-2, and Th2 cytokines). Our results showed that SPLUNC1 in the bronchoalveolar lavage fluid of OVA-challenged wild-type mice was significantly reduced (P < 0.05), which was negatively correlated with levels of lung eosinophilic inflammation. Moreover, SPLUNC1 KO mice demonstrated significantly higher numbers of eosinophils in the lung after OVA challenges than did wild-type mice. Alveolar macrophages isolated from OVA-challenged SPLUNC1 KO versus wild-type mice had higher concentrations of baseline eotaxin-2 that was amplified by LPS (a known risk factor for exacerbating asthma). Human recombinant SPLUNC1 protein was applied to alveolar macrophages to study the regulation of eotaxin-2 in the context of Th2 cytokine and LPS stimulation. Recombinant SPLUNC1 protein attenuated LPS-induced eotaxin-2 production in Th2 cytokine–pretreated murine macrophages. These findings demonstrate that SPLUNC1 inhibits airway eosinophilic inflammation in allergic mice, in part by reducing eotaxin-2 production in alveolar macrophages.
doi:10.1165/rcmb.2012-0064OC
PMCID: PMC3423460  PMID: 22499853
SPLUNC1; asthma; alveolar macrophage; Th2 cytokines; eotaxin-2
17.  Low Oxygen Saturation Target Range is Associated with Increased Incidence of Intermittent Hypoxemia 
The Journal of pediatrics  2012;161(6):1047-1052.
Objective
To test the hypothesis that preterm infants randomized to a low vs high O2 saturation target range have a higher incidence of intermittent hypoxemia.
Study design
A subcohort of 115 preterm infants with high resolution pulse oximetry enrolled in the Surfactant, Positive Pressure, and Oxygenation Randomized Trial were randomized to low (85%-89%) or high (91%-95%) O2 saturation target ranges. Oxygen saturation was monitored until 36 weeks postmenstrual age or until the infant was breathing room air without respiratory support for ≥72 hours.
Results
The low target O2 saturation group had a higher rate of intermittent hypoxemia (≤80% for ≥10 seconds and ≤3 minutes) prior to 12 days and beyond 57 days of life (P < .05). The duration shortened (P < .0001) and the severity increased (P < .0001) with increasing postnatal age with no differences between target saturation groups. The higher rate of intermittent hypoxemia events in the low target group was associated with a time interval between events of <1 minute.
Conclusion
A low O2 saturation target was associated with an increased rate of intermittent hypoxemia events that was dependent on postnatal age. The duration and severity of events was comparable between target groups. Further investigation is needed to assess the role of intermittent hypoxemia and their timing on neonatal morbidity.
doi:10.1016/j.jpeds.2012.05.046
PMCID: PMC3730286  PMID: 22738947
18.  Insect Nicotinic Receptor Agonists as Flea Adulticides in Small Animals 
Fleas are significant ectoparasites of small animals. They can be a severe irritant to animals and serve as a vector for a number of infectious diseases. In this article, we discuss the pharmacological characteristics of four insect nicotinic acetylcholine receptor (nAChR) agonists used as fleacides in dogs and cats, which include three neonicotinoids (imidacloprid, nitenpyram, and dinotefuran) and spinosad. Insect nAChR agonists are one of the most important new classes of insecticides, which are used to control sucking insects both on plants and on companion animals. These new compounds provide a new approach for practitioners to safely and effectively eliminate fleas.
doi:10.1111/j.1365-2885.2010.01160.x
PMCID: PMC3725634  PMID: 20646191
Fleas; neonicotinoids; spinosad; fleacides; nicotinic receptors
19.  Levamisole receptors: a second awakening 
Trends in Parasitology  2012;28(7):289-296.
Levamisole and pyrantel are old (1965) but useful anthelmintics that selectively activate nematode acetylcholine ion-channel receptors; they are used to treat roundworm infections in humans and animals. Interest in their actions has surged, giving rise to new knowledge and technical advances, including an ability to reconstitute receptors that reveal more details of modes of action/resistance. We now know that the receptors are plastic and may form diverse species-dependent subtypes of receptor with different sensitivities to individual cholinergic anthelmintics. Understanding the biology of the levamisole receptors is expected to inform other studies on anthelmintics (ivermectin and emodepside) that act on ion-channels.
doi:10.1016/j.pt.2012.04.003
PMCID: PMC3378725  PMID: 22607692
Levamisole; nematode; acetylcholine; ion channel receptor; subtypes; oocyte expression; anthelmintic; resistance
20.  Computational cloning of drug target genes of a parasitic nematode, Oesophagostomum dentatum 
BMC Genetics  2013;14:55.
Background
Gene identification and sequence determination are critical requirements for many biological, genomic, and bioinformatic studies. With the advent of next generation sequencing (NGS) technologies, such determinations are predominantly accomplished in silico for organisms for which the genome is known or for which there exists substantial gene sequence information. Without detailed genomic/gene information, in silico sequence determination is not straightforward, and full coding sequence determination typically involves time- and labor-intensive PCR-based amplification and cloning methods.
Results
An improved method was developed with which to determine full length gene coding sequences in silico using de novo assembly of RNA-Seq data. The scheme improves upon initial contigs through contig-to-gene identification by BLAST nearest–neighbor comparison, and through single-contig refinement by iterative-binning and -assembly of reads. Application of the iterative method produced the gene identification and full coding sequence for 9 of 12 genes and improved the sequence of 3 of the 12 genes targeted by benzimidazole, macrocyclic lactone, and nicotinic agonist classes of anthelminthic drugs in the swine nodular parasite Oesophagostomum dentatum. The approach improved upon the initial optimized assembly with Velvet that only identified full coding sequences for 2 genes.
Conclusions
Our reiterative methodology represents a simplified pipeline with which to determine longer gene sequences in silico from next generation sequence data for any nematode for which detailed genetic/gene information is lacking. The method significantly improved upon an initial Velvet assembly of RNA-Seq data that yielded only 2 full length sequences. The identified coding sequences for the 11 target genes enables further future examinations including: (i) the use of recombinant target protein in functional assays seeking a better understanding of the mechanism of drug resistance, and (ii) seeking comparative genomic and transcriptomic assessments between parasite isolates that exhibit varied drug sensitivities.
doi:10.1186/1471-2156-14-55
PMCID: PMC3689052  PMID: 23773280
Transcriptome; In silico sequence; Nematode; Anthelminthic; Drug resistance
21.  Mast Cells Protect against Airway Mycoplasma pneumoniae under Allergic Conditions 
Background
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.
Objective
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1111/j.1365-2222.2010.03488.x
PMCID: PMC2895012  PMID: 20345998
Asthma; host defense; innate immunity; mast cells; Mycoplasma pneumoniae
22.  IL-13 dampens human airway epithelial innate immunity through induction of IL-1 receptor–associated kinase M 
Background
Impaired airway mucosal immunity can contribute to increased respiratory tract infections in asthmatic patients, but the involved molecular mechanisms have not been fully clarified. Airway epithelial cells serve as the first line of respiratory mucosal defense to eliminate inhaled pathogens through various mechanisms, including Toll-like receptor (TLR) pathways. Our previous studies suggest that impaired TLR2 function in TH2 cytokine–exposed airways might decrease immune responses to pathogens and subsequently exacerbate allergic inflammation. IL-1 receptor–associated kinase M (IRAK-M) negatively regulates TLR signaling. However, IRAK-M expression in airway epithelium from asthmatic patients and its functions under a TH2 cytokine milieu remain unclear.
Objectives
We sought to evaluate the role of IRAK-M in IL-13–inhibited TLR2 signaling in human airway epithelial cells. Methods: We examined IRAK-M protein expression in epithelia from asthmatic patients versus that in normal airway epithelia. Moreover, IRAK-M regulation and function in modulating innate immunity (eg, TLR2 signaling) were investigated in cultured human airway epithelial cells with or without IL-13 stimulation.
Results
IRAK-M protein levels were increased in asthmatic airway epithelium. Furthermore, in primary human airway epithelial cells, IL-13 consistently upregulated IRAK-M expression, largely through activation of phosphoinositide 3-kinase pathway. Specifically, phosphoinositide 3-kinase activation led to c-Jun binding to human IRAK-M gene promoter and IRAK-M upregulation. Functionally, IL-13–induced IRAK-M suppressed airway epithelial TLR2 signaling activation (eg, TLR2 and human β-defensin 2), partly through inhibiting activation of nuclear factor κB.
Conclusions
Our data indicate that epithelial IRAK-M overexpression in TH2 cytokine–exposed airways inhibits TLR2 signaling, providing a novel mechanism for the increased susceptibility of infections in asthmatic patients.
doi:10.1016/j.jaci.2011.10.043
PMCID: PMC3348857  PMID: 22154382
IL-13; IL-1 receptor–associated kinase M; Toll-like receptor 2; airway epithelial cells
23.  Role of Arginase in Impairing Relaxation of Lung Parenchyma of Hyperoxia-Exposed Neonatal Rats 
Neonatology  2011;101(2):106-115.
Background
Prolonged exposure of immature lungs to hyperoxia contributes to neonatal lung injury and airway hyperreactivity. We have previously demonstrated that neonatal exposure of rat pups to ≥95% O2 impairs airway relaxation due to disruption of nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling. Objective: We now hypothesize that these impaired relaxation responses are secondary to hyperoxia-induced upregulation of arginase, which competes with NO synthase for L-arginine.
Methods
Rat pups were exposed to moderate neonatal hyperoxia (50% O2) or room air for 7 days from birth. In additional hyperoxic and room air groups, exogenous L-arginine (300 mg/kg/day i.p.) or arginase inhibitor (Nω-hydroxy-nor-arginine, 30 mg/kg/day i.p.) were administered daily. After 7 days, animals were anesthetized and sacrificed either for preparation of lung parenchymal strips or lung perfusion.
Results
In response to electrical field stimulation (EFS), bethanechol-preconstricted lung parenchymal strips from hyperoxic pups exhibited significantly reduced relaxation compared to room air controls. Supplementation of L-arginine or arginase blockade restored hyperoxia-induced impairment of relaxation. Expression of arginase I in airway epithelium was increased in response to hyperoxia but reduced by arginase blockade. Arginase activity was also significantly increased in hyperoxic lungs as compared to room air controls and reduced following arginase blockade. EFS-induced production of NO was decreased in hyperoxia-exposed airway smooth muscle and restored by arginase blockade.
Conclusion
These data suggest that NO-cGMP signaling is disrupted in neonatal rat pups exposed to even moderate hyperoxia due to increased arginase activity and consequent decreased bioavailability of the substrate L-arginine. We speculate that supplementation of arginine and/or inhibition of arginase may be a useful therapeutic tool to prevent or treat neonatal lung injury.
doi:10.1159/000329540
PMCID: PMC3223066  PMID: 21952491
Bronchodilation; Epithelium; L-Arginine; Nitric oxide production; Nω-hydroxy-nor-arginine; Lung strips; Oxygen
24.  Effect of Intermittent Hypercapnia on Respiratory Control in Rat Pups 
Neonatology  2009;97(2):117-123.
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.
doi:10.1159/000237222
PMCID: PMC3696363  PMID: 19752577
Hypoxia; Hypercapnia; Neonatal apnea; Chemoreceptors
25.  Clinical Expert Panel on Monitoring Potential Lung Toxicity of Inhaled Oligonucleotides: Consensus Points and Recommendations 
Nucleic Acid Therapeutics  2012;22(4):246-254.
Oligonucleotides (ONs) are an emerging class of drugs being developed for the treatment of a wide variety of diseases including the treatment of respiratory diseases by the inhalation route. As a class, their toxicity on human lungs has not been fully characterized, and predictive toxicity biomarkers have not been identified. To that end, identification of sensitive methods and biomarkers that can detect toxicity in humans before any long term and/or irreversible side effects occur would be helpful. In light of the public's greater interests, the Inhalation Subcommittee of the Oligonucleotide Safety Working Group (OSWG) held expert panel discussions focusing on the potential toxicity of inhaled ONs and assessing the strengths and weaknesses of different monitoring techniques for use during the clinical evaluation of inhaled ON candidates. This white paper summarizes the key discussions and captures the panelists' perspectives and recommendations which, we propose, could be used as a framework to guide both industry and regulatory scientists in future clinical research to characterize and monitor the short and long term lung response to inhaled ONs.
doi:10.1089/nat.2012.0345
PMCID: PMC3426204  PMID: 22809313

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