Impact of expression mode and timing of sample collection, relative to milk ejection, on human milk bacterial DNA profiles.

AIM: To investigate the impact of expression mode: electric breast pump or hand expression, and timing of sample collection: pre- and post-milk ejection on human milk (HM) bacterial DNA profiles. METHODS AND RESULTS: Three HM samples from the same breast were collected from 30 breastfeeding mothers: a pre-milk ejection pump-expressed sample (pre-pump), a post-milk ejection pump-expressed sample (post-pump) and a post-milk ejection hand-expressed sample (post-hand). Full-length 16S rRNA gene sequencing was used to assess milk bacterial DNA profiles. Bacterial profiles did not differ significantly based on mode of expression nor timing of sample collection. No significant differences were detected in the relative abundance of any OTUs based on expression condition (pre-pump/ post-pump and post-pump/post-hand) with univariate linear mixed-effects regression analyses (all P-values > 0·01; α = 0·01). Similarly, no difference in richness was observed between sample types (number of observed OTUs: post-pump/post-hand P = 0·13; pre-pump/post-pump P = 0. 45). CONCLUSION: Bacterial DNA profiles of HM did not differ according to either expression method or timing of sample collection. SIGNIFICANCE AND IMPACT OF THE STUDY: Hand or pump expression can be utilized to collect samples for microbiome studies. This has implications for the design of future HM microbiome studies.

DNA extraction method influences human milk bacterial profiles.

AIMS: To evaluate four DNA extraction methods to elucidate the most effective method for bacterial DNA recovery from human milk (HM). METHODS AND RESULTS: Human milk DNA was extracted using the following methods: (i) Qiagen MagAttract Microbial DNA Isolation Kit (kit QM), (ii) Norgen Milk Bacterial DNA Isolation Kit (kit NM), (iii) Qiagen MagAttract Microbiome DNA/RNA Isolation Kit (kit MM) and (iv) TRIzol LS Reagent (method LS). The full-length 16S rRNA gene was sequenced. Kits MM and method LS were unable to extract detectable levels of DNA in 9/11 samples. Detectable levels of DNA were recovered from all samples using kits NM (mean = 0·68 ng µl-1 ) and QM (mean = 0·55 ng µl-1 ). For kits NM and QM, the greatest number of reads were associated with Staphylococcus epidermidis, Streptococcus vestibularis, Propionibacterium acnes, Veillonella dispar and Rothia mucilaginosa. Contamination profiles varied substantially between kits, with one bacterial species detected in negative extraction controls generated with kit QM and six with kit NM. CONCLUSIONS: Kit QM is the most suitable of the kits tested for the extraction of bacterial DNA from human milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Choice of extraction method impacts the efficiency of bacterial DNA extraction from human milk and the resultant bacterial community profiles generated from these samples.

Effects of maternal dietary egg intake during early lactation on human milk ovalbumin concentration: a randomized controlled trial.

BACKGROUND: There is limited understanding of how maternal diet affects breastmilk food allergen concentrations, and whether exposure to allergens through this route influences the development of infant oral tolerance or sensitization. OBJECTIVE: To investigate how maternal dietary egg ingestion during early lactation influences egg protein (ovalbumin) levels detected in human breastmilk. METHODS: In a randomized controlled trial, women were allocated to a dietary group for the first six weeks of lactation: high-egg diet (> 4 eggs per week), low-egg diet (one-three eggs per week) or an egg-free diet. Breastmilk samples were collected at 2, 4 and 6 weeks of lactation for the measurement of ovalbumin. The permeability of the mammary epithelium was assessed by measuring the breastmilk sodium : potassium ratio. Egg-specific IgE and IgG4 were measured in infant plasma at 6 weeks, and prior to the introduction of egg in solids at 16 weeks. RESULTS: Average maternal egg ingestion was associated with breastmilk ovalbumin concentration. Specifically, for each additional egg ingested per week, there was an average 25% increase in ovalbumin concentration (95% CI: 5-48%, P = 0.01). Breastmilk ovalbumin concentrations were significantly higher in the 'high-egg' group (> 4 eggs per week) compared with the 'egg-free' group (P = 0.04). However, one-third of women had no breastmilk ovalbumin detected. No detectable associations were found between mammary epithelium permeability and breastmilk ovalbumin concentrations. Infant plasma egg-specific IgG4 levels were also positively associated with maternal egg ingestion, with an average 22% (95% CI: 3-45%) increase in infant egg-specific IgG4 levels per additional egg consumed per week (P = 0.02). CONCLUSIONS AND CLINICAL RELEVANCE: Increased maternal egg ingestion is associated with increased breastmilk ovalbumin, and markers of immune tolerance in infants. These results highlight the potential for maternal diet to benefit infant oral tolerance development during lactation.

Bioimpedance spectroscopy in the infant: effect of milk intake and extracellular fluid reservoirs on resistance measurements in term breastfed infants.

BACKGROUND/OBJECTIVES: Bioimpedance spectroscopy is an accurate non-invasive method for measuring body composition in adults, but in infants it requires further testing and validation. Of the few studies of bioimpedance conducted in infants, none have comprehensively investigated the effect of milk intake volume. This study assessed the effect of the milk intake, feed duration and the volume of the infant's stomach and bladder on the resistance values pre-/post-feed to establish the feasibility of using these values interchangeably during data collection. SUBJECTS/METHODS: Forty-eight breastfeeding infants were measured at 2, 5, 9 and/or 12 months (n=62 sessions) within 1-2 min before the start and after the end of breastfeed. Median (IQR) time between measurements was 24 (20.0-30.0) min. Resistance measurements at 0 and 50 kHz, and infinite frequency (R0, R50 and Rinf) and resistance of intracellular water (Ricw) were analysed with customised infant settings. Milk intake was measured by test weights. Free-water volumes and free-water change were determined from stomach and bladder volumes calculated from ultrasound images. RESULTS: Small pre-to-post-feed changes (median (IQR): R0 -3.7 (-14.8, 14.3); R50 0.3 (-10.4, 15.0); Rinf 2.8 (-13.3, 35.5); Ricw 20.8 (-98.1, 290.9)) were not significantly different from zero (R0: P=0.92; R50: P=0.48; Rinf: P=0.32; Ricw: P=0.097). No significant effect of milk intake or free-water change was detected. CONCLUSIONS: The lack of consistent change in resistance across a breastfeed provides flexibility in the timing of measurements of infants in the research setting, such that typically pre- and post-feed measures of resistance can be used interchangeably.

Novel feeding system to promote establishment of breastfeeds after preterm birth: a randomized controlled trial.

OBJECTIVE: We aimed to determine if a novel feeding system where milk only flowed when the preterm infant created a vacuum would influence time to full oral feeds, the length of stay (LOS) in hospital and breastfeeding at discharge. STUDY DESIGN: This was a randomized controlled trial in the tertiary neonatal intensive care unit at King Edward Memorial Hospital, Perth, Australia. Eligibility criteria were: preterm infants of gestational age 25 to 34 weeks receiving >75% human milk by gastric tube. Infants were randomly assigned to being fed with a novel teat (NT) or conventional teat (CT). Intention to treat analysis was performed. RESULT: Time to full suck feeds was not different between groups. LOS was shorter (mean: 2.5 days; P=0.026) and less formula was fed at discharge in the NT group (P=0.036). CONCLUSION: Use of a NT that releases milk when the infant applies vacuum while establishing breastfeeding reduces duration of hospitalization of preterm infants.

Atelectasis and survival after bronchoscopic lung volume reduction for COPD.

Bronchoscopic therapies to reduce lung volumes in chronic obstructive pulmonary disease are intended to avoid the risks associated with lung volume reduction surgery (LVRS) or to be used in patient groups in whom LVRS is not appropriate. Bronchoscopic lung volume reduction (BLVR) using endobronchial valves to target unilateral lobar occlusion can improve lung function and exercise capacity in patients with emphysema. The benefit is most pronounced in, though not confined to, patients where lobar atelectasis has occurred. Few data exist on their long-term outcome. 19 patients (16 males; mean±sd forced expiratory volume in 1 s 28.4±11.9% predicted) underwent BLVR between July 2002 and February 2004. Radiological atelectasis was observed in five patients. Survival data was available for all patients up to February 2010. None of the patients in whom atelectasis occurred died during follow-up, whereas eight out of 14 in the nonatelectasis group died (Chi-squared p=0.026). There was no significant difference between the groups at baseline in lung function, quality of life, exacerbation rate, exercise capacity (shuttle walk test or cycle ergometry) or computed tomography appearances, although body mass index was significantly higher in the atelectasis group (21.6±2.9 versus 28.4±2.9 kg·m(-2); p<0.001). The data in the present study suggest that atelectasis following BLVR is associated with a survival benefit that is not explained by baseline differences.

Mannose-binding lectin is present in the infected airway: a possible pulmonary defence mechanism.

BACKGROUND: Mannose-binding lectin (MBL) deficiency has been associated with infections of the respiratory tract and with increased disease severity in cystic fibrosis (CF). The mechanism is uncertain, and could relate either to systemic or local effects. The aim of this study was to determine, in a large cohort of children, whether MBL is present on the airway surface in health or disease. METHODS: Bronchoalveolar lavage (BAL) fluid from children with and without respiratory infection (some with underlying disease) was analysed for MBL and neutrophil elastase (NE). Levels were compared between groups, and correlations were examined with local and systemic inflammatory markers, infective organisms and load. RESULTS: 85 children were recruited to the study. MBL was absent in the lavage of all 7 children without lung infection but present in 62% (8/13) of those with acute pneumonia/pneumonitis, 23% (5/22) with recurrent respiratory tract infections, 17% (1/6) with primary ciliary dyskinesia and 8% (3/37) with CF (p<0.01). Children with acute pneumonia/pneumonitis had significantly higher levels than those in the other groups. There was no relationship with organisms cultured or systemic markers of inflammation, although in the group with detectable MBL in the BAL fluid, the levels correlated positively with levels of NE. CONCLUSIONS: MBL is undetectable in the non-infected airway but is present in a significant number of samples from children with lung infection. The levels found in the BAL fluid could be physiologically active and the protein may therefore be playing a role in host defence.

Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli.

Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.

Reproducibility of dynamically represented acoustic lung images from healthy individuals.

BACKGROUND AND AIM: Acoustic lung imaging offers a unique method for visualising the lung. This study was designed to demonstrate reproducibility of acoustic lung images recorded from healthy individuals at different time points and to assess intra- and inter-rater agreement in the assessment of dynamically represented acoustic lung images. METHODS: Recordings from 29 healthy volunteers were made on three separate occasions using vibration response imaging. Reproducibility was measured using quantitative, computerised assessment of vibration energy. Dynamically represented acoustic lung images were scored by six blinded raters. RESULTS: Quantitative measurement of acoustic recordings was highly reproducible with an intraclass correlation score of 0.86 (very good agreement). Intraclass correlations for inter-rater agreement and reproducibility were 0.61 (good agreement) and 0.86 (very good agreement), respectively. There was no significant difference found between the six raters at any time point. Raters ranged from 88% to 95% in their ability to identically evaluate the different features of the same image presented to them blinded on two separate occasions. CONCLUSION: Acoustic lung imaging is reproducible in healthy individuals. Graphic representation of lung images can be interpreted with a high degree of accuracy by the same and by different reviewers.

Sendai virus-mediated CFTR gene transfer to the airway epithelium.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Use of ultrasound to enhance nonviral lung gene transfer in vivo.

We have assessed if high-frequency ultrasound (US) can enhance nonviral gene transfer to the mouse lung. Cationic lipid GL67/pDNA, polyethylenimine (PEI)/pDNA and naked plasmid DNA (pDNA) were delivered via intranasal instillation, mixed with Optison microbubbles, and the animals were then exposed to 1 MHz US. Addition of Optison alone significantly reduced the transfection efficiency of all three gene transfer agents. US exposure did not increase GL67/pDNA or PEI/pDNA gene transfer compared to Optison-treated animals. However, it increased naked pDNA transfection efficiency by approximately 15-fold compared to Optison-treated animals, suggesting that despite ultrasound being attenuated by air in the lung, sufficient energy penetrates the tissue to increase gene transfer. US-induced lung haemorrhage, assessed histologically, increased with prolonged US exposure. The left lung was more affected than the right and this was mirrored by a lesser increase in naked pDNA gene transfer, in the left lung. The positive effect of US was dependent on Optison, as in its absence US did not increase naked pDNA transfection efficiency. We have thus established proof of principle that US can increase nonviral gene transfer, in the air-filled murine lung.

Computed tomography and cystic fibrosis: promises and problems.

Computed tomography (CT) has two potential roles in the evaluation of patients with cystic fibrosis (CF) lung disease: as a diagnostic test primarily for the detection of supervening complications and as a monitoring tool in clinical research. Interest in the latter role has gained momentum in the last 5 years because of two factors: (1) therapeutic options for CF lung disease are developing rapidly, hence the need for an outcome measure that can be applied in clinical intervention trials; and (2) it has become clear that traditional outcome measures such as pulmonary function tests are relatively insensitive to the early structural damage that occurs in CF. Several recent studies have shown that CT can be used as a potential surrogate outcome measure, although its suitability for this specific role is controversial and still under investigation. This review summarises current concepts relating to the research applications of CT in CF, with particular emphasis on the evidence supporting the use of CT as a surrogate outcome measure in clinical trials.

Gene therapy progress and prospects: cystic fibrosis.

Our first review on progress and prospects in cystic fibrosis (CF) gene therapy was published in this series in October 2002. We now summarize the progress made since then and comment on the prospects for CF gene therapy over the next couple of years. Three clinical trials have been carried out, further supporting the proof-of-principle that gene transfer to the airway epithelium is feasible. Developments in viral and non-viral vectors, as well as recent alternative strategies such as gene repair, trans-splicing and stem cell therapy will be reviewed.

Intravenously administered oligonucleotides can be delivered to conducting airway epithelium via the bronchial circulation.

Topical gene transfer to the airways of cystic fibrosis (CF) patients has been inefficient, partly due to extracellular barriers such as sputum. In an attempt to circumvent these, we assessed whether airway epithelial cells can be transfected by intravenous (i.v.) administration of liposome-complexed or "naked" oligonucleotides (ODNs). The conducting airways are the likely target for CF therapy and are supplied by the bronchial circulation. Consequently, we assessed ODN transfer in the mouse trachea and main bronchi as these are supplied by the bronchial circulation. Liposome-protamine-DNA (LPD) complexes were detected in the bronchial circulation but did not transfect conducting airway epithelial cells, even in the presence of microvascular leakage. In contrast, 'naked' ODNs were delivered to 17% (inter-quartile range (IQR) 10-34%) and 35% (IQR 24-59%) of epithelial cells when injected at 500 microg/animal, without and with microvascular leakage, respectively. Two types of nuclear signal were observed; punctate in cells throughout the airways (3%, IQR 2-6%, and 6%, IQR 4-7%, of cells when delivered without and with microvascular leakage, respectively) and diffuse in a small number of epithelial cells in the proximal trachea. ODNs may be relevant to CF in a variety of ways and these data suggest one way towards implementing their use.

Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo.

We have assessed whether magnetic forces (magnetofection) can enhance non-viral gene transfer to the airways. TransMAG(PEI), a superparamagnetic particle was coupled to Lipofectamine 2000 or cationic lipid 67 (GL67)/plasmid DNA (pDNA) liposome complexes. In vitro transfection with these formulations resulted in approximately 300- and 30-fold increase in reporter gene expression, respectively, after exposure to a magnetic field, but only at suboptimal pDNA concentrations. Because GL67 has been formulated for in vivo use, we next assessed TransMAG(PEI) in the murine nasal epithelium in vivo, and compared this to naked pDNA. At the concentrations required for in vivo experiments, precipitation of magnetic complexes was seen. After extensive optimization, addition of non-precipitated magnetic particles resulted in approximately seven- and 90-fold decrease in gene expression for naked pDNA and GL67/pDNA liposome complexes, respectively, compared to non-magnetic particles. Thus, whereas exposure to a magnetic field improved in vitro transfection efficiency, translation to the in vivo setting remains difficult.

Effect and duration of lung volume reduction surgery: mid-term results of the Brompton trial.

UNLABELLED: Although many studies have reported improvement in lung function following LVRS, the magnitude of improvement and subsequent decline has not been evaluated against medical therapy after the second year. METHODS: Existing pulmonary function records were collapsed for ech participant since randomisation from Brompton LVRS trial cohort. Longitudinal data analysis was used to profile th history of medically treated patients and the effect of LVRS. RESULTS: Pulmonary function results were collated from survivors over a median of 25 (17 to 39) months. The estimated immediate increase in mean FEV1, following surgery was +0.2591 (0.179, 0.339), with a rate of change of -0.0051 (-0.009, -0.001) per month compared to medical therapy (p < 0.001). The changes in the secondary outcome measures (LVRS compared to medical therapy) were an increase in FVC (p = 0.004), decrease in RV (p < 0.001) and TLC (p < 0.001), with differences that were maintained over time. The initial reduction in RV/TLC ration was sustained (p < 0.001), but the estimated initial increase in peak flow was accompanied by a gradual decline that was not statistically significant (p = 0.062). KCOc showed no immediate change, but there was a gradual sustained increase with time (p = 0.009). Mean oxygen saturations improved and continued to do so compared to patients on medical therapy (p = 0.001). CONCLUSIONS: The immediate increase in FEV1 is not sustained, although the mechanical improvements of LVRS on increasing FVC, reducing both the RV and RV/TLC ratio, appear to be maintained. The important benefits of LVRS may be the gradual and sustained increase in transfer factor accompanied by improved oxygen saturations.