Cephalosporin-3'-Diazeniumdiolate NO Donor Prodrug PYRRO-C3D Enhances Azithromycin Susceptibility of Nontypeable Haemophilus influenzae Biofilms.

PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking ß-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability (P < 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed (P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.

Primary ciliary dyskinesia ciliated airway cells show increased susceptibility to Haemophilus influenzae biofilm formation.

Non-typeable Haemophilus influenzae (NTHi) is the most common pathogen in primary ciliary dyskinesia (PCD) patients. We hypothesised that abnormal ciliary motility and low airway nitric oxide (NO) levels on airway epithelial cells from PCD patients might be permissive for NTHi colonisation and biofilm development.We used a primary epithelial cell co-culture model to investigate NTHi infection. Primary airway epithelial cells from PCD and non-PCD patients were differentiated to ciliation using an air-liquid interface culture and then co-cultured with NTHi.NTHi adherence was greater on PCD epithelial cells compared to non-PCD cells (p<0.05) and the distribution of NTHi on PCD epithelium showed more aggregated NTHi in biofilms (p<0.001). Apart from defective ciliary motility, PCD cells did not significantly differ from non-PCD epithelial cells in the degree of ciliation and epithelial integrity or in cytokine, LL-37 and NO production. Treatment of PCD epithelia using exogenous NO and antibiotic significantly reduced NTHi viability in biofilms compared with antibiotic treatment alone.Impaired ciliary function was the primary defect in PCD airway epithelium underlying susceptibility to NTHi biofilm development compared with non-PCD epithelium. Although NO responses were similar, use of targeted NO with antibiotics enhanced killing of NTHi in biofilms, suggesting a novel therapeutic approach.

Respiratory consequences of late preterm birth.

In developed countries most preterm births occur between 34 and 37 weeks' gestation. Deliveries during this 'late preterm' period are increasing and, since even mild prematurity is now recognised to be associated with adverse health outcomes, this presents healthcare challenges. Respiratory problems associated with late preterm birth include neonatal respiratory distress, severe RSV infection and childhood wheezing. Late preterm birth prematurely interrupts in utero lung development and is associated with maternal and early life factors which adversely affect the developing respiratory system. This review considers 1) mechanisms underlying the association between late preterm birth and impaired respiratory development, 2) respiratory morbidity associated with late preterm birth, particularly long-term outcomes, and 3) interventions which might protect respiratory development by addressing risk factors affecting the late preterm population, including maternal smoking, early life growth restriction and vulnerability to viral infection.

Nasal nitric oxide screening for primary ciliary dyskinesia: systematic review and meta-analysis.

Nasal nitric oxide (nNO) concentrations are low in patients with primary ciliary dyskinesia (PCD) providing a noninvasive screening test. We conducted a systematic review of the literature to examine the utility of nNO in screening for PCD, in particular 1) different respiratory manoeuvres during sampling (velum closure, tidal breathing, etc.), 2) accuracy in screening young/uncooperative children, 3) stationary versus portable analysers, and 4) nNO in "atypical" PCD. 96 papers were assessed according to modified PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) criteria and 22 were included in this review. Meta-analysis of 11 studies comparing nNO during a velum closure breath hold gave a mean±SD nNO of 19.4±18.6 nL·min(-1) in PCD (n = 478) and 265.0±118.9 nL·min(-1) in healthy controls (n = 338). Weighted mean difference for PCD versus healthy controls was 231.1 nL·min(-1) (95% CI 193.3-268.9; n = 338) and 114.1 nL·min(-1) (95% CI 101.5-126.8; n = 415) for PCD versus cystic fibrosis. Five studies of nNO measurement during tidal breathing demonstrated that this is an acceptable manoeuvre in young children where velum closure is not possible, but the discriminatory value was reduced. Four small studies of portable NO analysers suggest these are reliable tools for screening for PCD. However, nNO must be interpreted alongside clinical suspicion. Future studies should focus on standardising sampling techniques and reporting.

A BEAT-PCD consensus statement: a core outcome set for pulmonary disease interventions in primary ciliary dyskinesia.

Background: Consistent use of reliable and clinically appropriate outcome measures is a priority for clinical trials, with clear definitions to allow comparability. We aimed to develop a core outcome set (COS) for pulmonary disease interventions in primary ciliary dyskinesia (PCD). Methods: A multidisciplinary international PCD expert panel was set up. A list of outcomes was created based on published literature. Using a modified three-round e-Delphi technique, the panel was asked to decide on relevant end-points related to pulmonary disease interventions and how they should be reported. First, inclusion of an outcome in the COS was determined. Second, the minimum information that should be reported per outcome. The third round finalised statements. Consensus was defined as ≥80% agreement among experts. Results: During the first round, experts reached consensus on four out of 24 outcomes to be included in the COS. Five additional outcomes were discussed in subsequent rounds for their use in different subsettings. Consensus on standardised methods of reporting for the COS was reached. Spirometry, health-related quality-of-life scores, microbiology and exacerbations were included in the final COS. Conclusion: This expert consensus resulted in a COS for clinical trials on pulmonary health among people with PCD.

The relationship between maternal adiposity and infant weight gain, and childhood wheeze and atopy.

BACKGROUND: Obesity and asthma have increased in westernised countries. Maternal obesity may increase childhood asthma risk. If this relation is causal, it may be mediated through factors associated with maternal adiposity, such as fetal development, pregnancy complications or infant adiposity. We investigated the relationships of maternal body mass index (BMI) and fat mass with childhood wheeze, and examined the influences of infant weight gain and childhood obesity. METHODS: Maternal prepregnancy BMI and estimated fat mass (from skinfold thicknesses) were related to asthma, wheeze and atopy in 940 children. Transient or persistent/late wheeze was classified using questionnaire data collected at ages 6, 12, 24 and 36 months and 6 years. At 6 years, skin-prick testing was conducted and exhaled nitric oxide and spirometry measured. Infant adiposity gain was calculated from skinfold thickness at birth and 6 months. RESULTS: Greater maternal BMI and fat mass were associated with increased childhood wheeze (relative risk (RR) 1.08 per 5 kg/m(2), p=0.006; RR 1.09 per 10 kg, p=0.003); these reflected associations with transient wheeze (RR 1.11, p=0.003; RR 1.13, p=0.002, respectively), but not with persistent wheeze or asthma. Infant adiposity gain was associated with persistent wheeze, but not significantly. Adjusting for infant adiposity gain or BMI at 3 or 6 years did not reduce the association between maternal adiposity and transient wheeze. Maternal adiposity was not associated with offspring atopy, exhaled nitric oxide, or spirometry. DISCUSSION: Greater maternal adiposity is associated with transient wheeze but not asthma or atopy, suggesting effects upon airway structure/function but not allergic predisposition.

Validation of the Paediatric Food Allergy Quality of Life Questionnaire (PFA-QL).

BACKGROUND: The Paediatric Food Allergy Quality of Life Questionnaire (PFA-QL) was the first tool to be developed for assessing health-related quality of life (QoL) in children with food allergy. It has been used in a number of published studies, but has not been validated. OBJECTIVE: The aim of the current study was to validate child (PFA-QL) and parent-proxy (PFA-QL-PF) versions of the scale in a specialist allergy clinic and in parents of children with food allergy. METHODS: For the clinic sample, a generic QoL scale (PedsQL) and the PFA-QL were completed by 103 children (age 6-16 yrs) with peanut or tree nut allergy; test-retest reliability of the PFA-QL was tested in 50 stable patients. For the non-clinical sample, 756 parents of food allergic children completed the PFA-QL-PF, the Child Health Questionnaire (CHQ-PF50), Food Allergy Quality of Life Parental Burden Scale (FAQL-PB) and a Food Allergy Impact Measure. RESULTS: The PFA-QL and PFA-QL-PF had good internal consistency (α's of 0.77-0.82), and there was moderate-to-good agreement between the generic- and disease-specific questionnaires. The PFA-QL was stable over time in the clinic sample, and in both samples, girls were reported to have poorer QoL than boys. CONCLUSIONS: The PFA-QL and PFA-QL-PF are reliable and valid scales for use in both clinical and non-clinical populations. Unlike other available tools, they were developed and validated in the UK and thus provide a culture-specific choice for research, clinical trials and clinical practice in the UK. Validation in other countries is now needed.

Management of primary ciliary dyskinesia in European children: recommendations and clinical practice.

The European Respiratory Society Task Force on primary ciliary dyskinesia (PCD) in children recently published recommendations for diagnosis and management. This paper compares these recommendations with current clinical practice in Europe. Questionnaires were returned by 194 paediatric respiratory centres caring for PCD patients in 26 countries. In most countries, PCD care was not centralised, with a median (interquartile range) of 4 (2-9) patients treated per centre. Overall, 90% of centres had access to nasal or bronchial mucosal biopsy. Samples were analysed by electron microscopy (77%) and ciliary function tests (57%). Nasal nitric oxide was used for screening in 46% of centres and saccharine tests in 36%. Treatment approaches varied widely, both within and between countries. European region, size of centre and the country's general government expenditure on health partly defined availability of advanced diagnostic tests and choice of treatments. In conclusion, we found substantial heterogeneity in management of PCD within and between countries, and poor concordance with current recommendations. This demonstrates how essential it is to standardise management and decrease inequality between countries. Our results also demonstrate the urgent need for research: to simplify PCD diagnosis, to understand the natural history and to test the effectiveness of interventions.

Maternal plasma phosphatidylcholine fatty acids and atopy and wheeze in the offspring at age of 6 years.

Variation in exposure to polyunsaturated fatty acids (PUFAs) might influence the development of atopy, asthma, and wheeze. This study aimed to determine whether differences in PUFA concentrations in maternal plasma phosphatidylcholine are associated with the risk of childhood wheeze or atopy. For 865 term-born children, we measured phosphatidylcholine fatty acid composition in maternal plasma collected at 34 weeks' gestation. Wheezing was classified using questionnaires at 6, 12, 24, and 36 months and 6 years. At age of 6 years, the children underwent skin prick testing, fractional exhaled nitric oxide (FENO) measurement, and spirometry. Maternal n-6 fatty acids and the ratio of n-3 to n-6 fatty acids were not associated with childhood wheeze. However, higher maternal eicosapentaenoic acid, docosahexaenoic acid, and total n-3 fatty acids were associated with reduced risk of non-atopic persistent/late wheeze (RR 0.57, 0.67 and 0.69, resp. P = 0.01, 0.015, and 0.021, resp.). Maternal arachidonic acid was positively associated with FENO (P = 0.024). A higher ratio of linoleic acid to its unsaturated metabolic products was associated with reduced risk of skin sensitisation (RR 0.82, P = 0.013). These associations provide some support for the hypothesis that variation in exposure to n-6 and n-3 fatty acids during pregnancy influences the risk of childhood wheeze and atopy.

Patterns of fetal and infant growth are related to atopy and wheezing disorders at age 3 years.

BACKGROUND: Little is known about whether patterns of early growth are associated with altered respiratory and immune development. This study relates prenatal and infant growth patterns to wheeze and atopy at age 3 years. METHODS: Birth weight and length were measured in 1548 children born at term. Conditional fetal head and abdominal circumference growth velocities were calculated from antenatal ultrasound measurements. Conditional postnatal growth velocities were calculated from infant weight, length and adiposity data. Measures of size and conditional growth were related to parentally-reported infant and early childhood wheeze and to atopic status at age 3 years. RESULTS: The risk of atopy increased by 46% per SD increase in abdominal circumference growth velocity from 11 to 19 weeks gestation but by 20% per SD decrease in abdominal growth velocity from 19 to 34 weeks (p=0.007 and p=0.011, respectively). The risk of atopic wheeze increased by 20% per SD decrease in 19-34-week abdominal growth (p=0.046). The risk of non-atopic wheeze increased by 10% per SD decrease in 11-19-week head circumference growth. Greater relative infant weight and adiposity gains were associated with both atopic and non-atopic wheeze. CONCLUSIONS: A rapid growth trajectory during 11-19 weeks gestation followed by late gestation growth faltering is associated with atopy, suggesting that influences affecting fetal growth may also alter immune development. A lower early fetal growth trajectory is associated with non-atopic wheeze, possibly reflecting an association with smaller airways. An association between postnatal adiposity gain and wheeze may partly reflect prenatal influences that cause fetal growth to falter but are then followed by postnatal adiposity gain.

Management of nut allergy influences quality of life and anxiety in children and their mothers.

Nut allergy is known to impact on the quality of life (QoL) and anxiety of both the allergic child and their parents, but little is known about how the management of food allergy is associated with these variables. To investigate the impact of nut allergy on QoL and anxiety in mothers and children with nut allergy in order to identify management strategies that may influence these factors. Forty-one nut allergic children (age 6-16 yrs) and their mothers completed questionnaires to assess maternal and children's QoL (PedsQL, WHOQOL-BREF, FAQL-PB), anxiety (SCAS, STAI) and perceived stress scale (PSS). Children also completed a nut allergy specific QoL questionnaire. Demographic data, details of previous reactions, test results and management plans were collected using parent-report questionnaires and hospital notes. Children with nut allergy had poorer emotional (p = 0.004), social (p = 0.043), and psychological (p = 0.006) QoL compared to healthy normative data. Maternal and child QoL and anxiety were not influenced by the severity of previous reactions. Mother and child reported lower anxiety (p = 0.043 and p < 0.001 respectively) when the child was prescribed an epinephrine auto-injector. Anxiety was not associated with whether the child carried the auto-injector or whether they strictly avoided traces of nuts in foods. Prescribing auto-injectors is associated with reduced anxiety for food allergic children and their mothers, but is not associated with improved adherence with medical management or reduced risk-taking behavior.

The effect of digestion and pH on the allergenicity of kiwifruit proteins.

It is suggested that patients with oral allergy syndrome (OAS) respond to pepsin-sensitive allergens, and systemic reactors identify pepsin-resistant allergens. We sought to assess the digestibility of kiwifruit proteins in simulated gastric fluid (SGF), and to compare the immunogenicity of the digests in patients with isolated oral and systemic reactions to kiwifruit. In addition, the effect of pH on digestibility of kiwifruit proteins was investigated. The in vitro resistance of kiwifruit proteins to digestion was determined using SGF. G-immunoglobulin (IgE) binding to digested proteins was investigated by Western blotting using sera from children and adults (aged 5-72 yr) with systemic reactions and patients with isolated oral symptoms. To determine whether pH conditions influence digestion of kiwifruit extracts, digestion at pHs 1.5-7 were compared by SDS-PAGE. Patients with systemic reactions showed IgE binding to digestion-resistant allergens, but patients with oral symptoms reacted only to digestion-labile allergens. An increase in pH from 1.5 to 2.5 significantly reduced pepsin breakdown of kiwifruit allergens. Immunoreactive digested protein fragments were detectable by immunoblot but not Coomassie stain. This study confirms a difference in the lability of food allergens recognized by patients with systemic reactions and those with OAS. Pepsin digestion of kiwifruit proteins was impaired by hypoacidic conditions suggesting that patients with hypoacidic gastric conditions are at increased risk of systemic absorption of allergens. The data indicate that commonly used methods for predicting allergenicity of novel proteins using Coomassie stains may be flawed.

Maternal body mass index: Relation with infant respiratory symptoms and infections.

BACKGROUND: Maternal obesity is increasingly prevalent in many westernized countries. Many studies report associations between maternal obesity and childhood wheeze or asthma but few have considered maternal obesity in relation to respiratory infections or symptoms other than wheeze during infancy. This study assesses the relationship between maternal BMI and reported wheeze, cough and respiratory infections during the first year of life. METHODS: In 2799 mother-child pairs, we examined the relations between maternal pre-pregnancy BMI and pregnancy weight gain and reported offspring wheeze, prolonged cough, lower respiratory tract infection, croup, and ear infection before age 1 year, along with reported diarrhea or vomiting. Maternally reported paternal BMI was included in the models as a proxy for unmeasured confounding by shared familial factors. RESULTS: Higher maternal BMI was associated with increased risks of offspring wheeze, prolonged cough and lower respiratory tract infection (relative risks (95%CI) per 5 kg/m2 1.09 (1.05-1.13), 1.09 (1.03-1.14), and 1.13 (1.07-1.20), respectively). These associations remained after adjusting for maternally reported paternal BMI. No associations were found with croup, ear infection, or diarrhea or vomiting. Pregnancy weight gain was not associated with any of the offspring symptoms or illnesses. DISCUSSION: Higher maternal BMI is associated with increased risk of wheeze, cough, and maternally reported lower respiratory tract infection in infancy. These associations were independent of maternally reported paternal BMI. These observations might be explained by intrauterine effects of maternal obesity upon respiratory or immune development.

Prenatal development is linked to bronchial reactivity: epidemiological and animal model evidence.

Chronic cardiorespiratory disease is associated with low birthweight suggesting the importance of the developmental environment. Prenatal factors affecting fetal growth are believed important, but the underlying mechanisms are unknown. The influence of developmental programming on bronchial hyperreactivity is investigated in an animal model and evidence for comparable associations is sought in humans. Pregnant Wistar rats were fed either control or protein-restricted diets throughout pregnancy. Bronchoconstrictor responses were recorded from offspring bronchial segments. Morphometric analysis of paraffin-embedded lung sections was conducted. In a human mother-child cohort ultrasound measurements of fetal growth were related to bronchial hyperreactivity, measured at age six years using methacholine. Protein-restricted rats' offspring demonstrated greater bronchoconstriction than controls. Airway structure was not altered. Children with lesser abdominal circumference growth during 11-19 weeks' gestation had greater bronchial hyperreactivity than those with more rapid abdominal growth. Imbalanced maternal nutrition during pregnancy results in offspring bronchial hyperreactivity. Prenatal environmental influences might play a comparable role in humans.

Validation of novel wheeze phenotypes using longitudinal airway function and atopic sensitization data in the first 6 years of life: evidence from the Southampton Women's survey.

BACKGROUND: In 1995 the Tucson Children's Respiratory Study (TCRS) identified clinically distinct phenotypes amongst early wheezers; the Avon Longitudinal Study of Parents And Children (ALSPAC) has recently re-examined these. OBJECTIVES: To validate statistically derived ALSPAC phenotypes in the Southampton Women's Survey (SWS) using infant and 6-year lung function, and allergic sensitization at 1, 3, and 6 years, comparing these with TCRS phenotypes. METHODS: Complete 6-year follow-up data were available for 926 children, selected from 1,973 infants born to 12,579 women characterized pre-conception. Ninety-five children had V'maxFRC and FEV0.4 measured age 5-14 weeks using rapid compression/raised volume techniques. At 6 years we performed spirometry (n = 791), fractional exhaled nitric oxide (FeNO, n = 589) and methacholine challenge (n = 234). Skin prick testing was performed at 12m, 3 and 6 years (n = 1,494, 1,255, 699, respectively). Using wheeze status questionnaire data at 6m, 12m, 2, 3 and 6 years we classified children into TCRS (never, transient early, persistent, late-onset) and ALSPAC based groups (never, early, transient, intermediate-onset, late-onset, persistent). RESULTS: Amongst ALSPAC groups, persistent and late-onset wheeze were associated with atopy at 3 and 6 years, whilst intermediate-onset wheeze showed earlier atopic association at 1 year; all three were associated with FeNO at 6 years. Persistent wheezers had lower infant (V'maxFRC P < 0.05) and 6-year lung function (FEV1, FEV1/FVC, and FEF(25-75), P < 0.05), whilst late and intermediate-onset wheezers showed no lung function deficits. Transient wheezers were non-atopic but showed persistent lung function deficits (V'maxFRC in infancy, FEV1 and FEF(25-75) at 6 years, all P < 0.05). Those who wheezed only in the first year (early phenotype) showed no lung function deficits. No associations were seen with 6 years bronchial hyper-responsiveness or infancy FEV0.4. CONCLUSION: SWS cohort data validates the statistically derived ALSPAC six-class model. In particular, lung function and atopy successfully differentiate persistent, late-onset and intermediate-onset wheeze, whilst the Tucson "transient early" wheeze phenotype can be sub-classified into groups that reflect early lung function. Since the 4-class model fails to adequately differentiate phenotypes based on lung function and atopy, we propose that strong consideration be given to using the 6-class paradigm for longitudinal outcome work in wheezing with onset in early life.

Exhaled nitric oxide monitoring does not reduce exacerbation frequency or inhaled corticosteroid dose in paediatric asthma: a randomised controlled trial.

INTRODUCTION: Inhaled corticosteroid therapy (ICS) for asthma is currently modified according to symptoms and lung function. Fractional exhaled nitric oxide (FENO) has been demonstrated to be a non-invasive marker of eosinophilic inflammation. Studies of FENO-driven asthma management show variable success. Objectives: This study aimed to evaluate whether monitoring FENO can improve outpatient management of children with moderate to severe asthma using a pragmatic design. METHODS: Children aged 6­17 years with moderate to severe asthma were recruited. Their asthma was stabilised before randomisation to FENO-driven therapy or to a standard management group where therapy was driven by conventional markers of asthma control. ICS or long-acting bronchodilator therapies were altered according to FENO levels in combination with reported symptoms in the FENO group. Participants were assessed 2 monthly for 12 months. ICS dose and exacerbation frequency change were compared between groups in an intention to treat analysis. RESULTS: Ninety children were randomised. No difference was found between the two groups in either change in corticosteroid dose or exacerbation frequency. Results were similar in a planned secondary analysis of atopic asthmatics. CONCLUSION: FENO-guided ICS titration does not appear to reduce corticosteroid usage or exacerbation frequency in paediatric outpatients with moderate to severe asthma. This may reflect limitations in FENO-driven management algorithms, as there are now concerns that FENO levels relate to atopy as much as they relate to asthma control.

Comparison of the allergenicity of Actinidia deliciosa (kiwi fruit) and Actinidia chinensis (gold kiwi).

Actinidia chinensis (gold kiwi) is a newly available fruit which has been shown to have in vitro immunoglobulin E (IgE) cross-reactivity with green kiwi. This is the first study to investigate clinical reactivity of gold kiwi. Five patients clinically allergic to green kiwi were investigated by skin test and double-blind placebo controlled food challenge (DBPCFC) with gold kiwi fruit. IgE-binding patterns of individual sera from the five challenged patients and a pool of sera from a further nine patients with kiwi allergy were compared in the two fruits by Western blotting. Cross reactivity of proteins in the two fruits was assessed by inhibition of immunoblots and by IgE enzyme-linked immunosorbent assay (ELISA) inhibition. Four of the five patients had a positive DBPCFC to gold kiwi. Western blotting showed marked differences in the allergen patterns of green and gold kiwi. However, inhibition of the immunoblots and ELISA assay reveals extensive inhibition of IgE binding to proteins in each fruit by the alternative species. Gold kiwi fruit is allergenic and patients allergic to green kiwi are at risk of reacting to the gold kiwi fruit. Despite having different protein profiles and IgE-binding patterns, the two species have proteins that extensively cross-inhibit the binding to IgE.