Novel expression of Haemonchus contortus vaccine candidate aminopeptidase H11 using the free-living nematode Caenorhabditis elegans

With the problem of parasitic nematode drug resistance increasing, vaccine development offers an alternative sustainable control approach. For some parasitic nematodes, native extracts enriched for specific proteins are highly protective. However, recombinant forms of these proteins have failed to replicate this protection. This is thought to be due to differences in glycosylation and/or conformation between native and recombinant proteins. We have exploited the free-living nematode Caenorhabditis elegans to examine its suitability as an alternative system for recombinant expression of parasitic nematode vaccine candidates. We focussed on Haemonchus contortus aminopeptidase H11 glycoprotein, which is enriched in a gut membrane fraction capable of inducing significant protection against this important ovine gastrointestinal nematode. We show that H. contortus H11 expressed in C. elegans is enzymatically active and MALDI mass spectrometry identifies similar di- and tri-fucosylated structures to those on native H11, with fucose at the 3- and/or 6-positions of the proximal GlcNAc. Some glycan structural differences were observed, such as lack of LDNF. Serum antibody to native H11 binds to C. elegans recombinant H11 and most of the antibody to rH11 or native H11 is directed to glycan moieties. Despite these similarities, no reduction in worm burden or faecal egg count was observed following immunisation of sheep with C. elegans-expressed recombinant H11 protein. The findings suggest that the di- and tri-fucosylated N-glycans expressed on rH11 do not contribute to the protective effect of H11 and that additional components present in native H11-enriched extract are likely required for enhancing the antibody response necessary for protection

The use of Wonnolas in controlling gastrointestinal nematode infections in sheep under traditional grazing management in Indonesia

Blocks containing 3% phenothiazine in solidified molasses (Wonnolas, Animeal Australia Ltd .) were tested to control gastrointestinal nematode infections and the effect on mineral status in sheep in villages in Cirebon, Indonesia. Two hundred and thirteen Javanese Thin Tail sheep which were grazed during the day and housed at night were divided into two groups on the basis of location . One group was allowed in an access to Wonnolas blocks when penned for 24 weeks while the other group remained untreated. The bodyweight was recorded and faecal samples were collected for egg counts and larval culture every four weeks. Sera and saliva samples were collected from 20 adult sheep in each group at weeks 0, 12 and 24 for mineral analysis by AAS and colorimetry. The results indicated that the mean egg counts of the treated group decreased from 576 epg to 123 epg and the percentage of sheep producing viable larvae decreased from 50% to 24%. In contrast egg counts of the control group increased from 768 epg to 4,840 epg and the percentage of sheep producing viable larvae increased from 65% to 84% over the same period . In the treated group the number of Haenronchus larvae declined significantly (36% to 6 %) and at the end of the trial Tiichostrongylus larvae predominanted in larval cultures (80%) . Mineral analysis revealed deficiencies in sodium and copper, low levels of zinc and normal levels of potassium, calcium, magnesium and phosphorus . Wormolas had a significant effect on sodium and zinc status but not on copper although sufficient levels of this element were available. Comparison of bodyweight gains showed a significantly (P0.05) higher rate of increase in the treated animals

Use of novel DNA methylation signatures to distinguish between human airway structural cell types

INTRODUCTION: Chronic inflammatory and fibrotic lung diseases like asthma, COPD and pulmonary fibrosis are characterised by modified phenotype of the airway structural cells. Airway walls are comprised of a robust epithelial layer that lines the lumen followed by the basement membrane, submucosa predominantly composed of fibroblasts and finally enveloped by a bulk of smooth muscle cells that determine the relaxation and constriction of the airways. The phenotype of airway structural cells is determined by epigenetic alterations such as DNA methylation, which alters the activation status of a range of important inflammatory and remodelling genes. Here we determined if airway structural cells (Epithelial cells, fibroblasts and smooth muscle cells) have different DNA methylome signatures that can be used to distinguish between them. This will offer a reference standard for identifying cell type specific DNA methylation changes induced by various inflammatory stimuli. EXPERIMENTAL METHODS: Illumina Human Methylation 450K Beadchip (HM450K) was used to perform genome-wide methylome screening on 17 bronchial fibroblast (BrF), 23 lung parenchymal fibroblast (LgF), 17 airway epithelial cell (Ep) and 6 airway smooth muscle cell (ASM) samples isolated from healthy individuals. The data was normalised using funtoonorm, a specialised algorithm in R developed for multiple tissue types. R packages minfi, limma and DMRcate was used for CpG site exclusion and identification of significant differentially methylated regions (DMR) specific to each of the four cell types. RESULTS AND DISCUSSION: Epithelial cells distinctly separated from other lung cells (791 DMR). LgF, BrF and ASM had 13, 10 and 1 signature DMR respectively. Despite close anatomical proximity, ASM and BrF displayed 2 DMR when compared to each other. Interestingly, fibroblasts obtained from airway showed 6 DMR in comparison to those obtained from lung parenchyma, suggesting that the same cell type obtained from different parts of the lung can have significantly different methylation patterns that might lead to phenotypic differences. CONCLUSION: We have identified cell and tissue specific methylation signatures which can be used to differentiate between different types of airway structural cells. The airway epithelial cells showed the greatest separation from other airway structural cells. The Bronchial fibroblasts varied minimally from airway smooth muscle cells despite its significant separation from airway epithelial cells and parenchymal fibroblasts

The pharmacokinetics and toxicity of morning vs. evening tobramycin dosing for pulmonary exacerbations of cystic fibrosis:A randomised comparison

AbstractBackgroundCircadian variation in renal toxicity of aminoglycosides has been demonstrated in animal and human studies. People with CF are frequently prescribed aminoglycosides. Altered pharmacokinetics of aminoglycosides are predictive of toxicity.AimTo investigate whether the time of day of aminoglycoside administration modulates renal excretion of tobramycin and toxicity in children with CF. To determine whether circadian rhythms are disrupted in children with CF during hospital admission.MethodsChildren (age 5–18years) with CF scheduled for tobramycin therapy were randomly allocated to receive tobramycin at 0800 or 2000h. Serum tobramycin levels were drawn at 1h and between 3.5 and 5h post-infusion between days 5 and 9 of therapy. Melatonin levels were measured serially at intervals from 1800h in the evening until 1200h on the next day. Circadian rhythm was categorised as normal when dim light melatonin onset was demonstrated between 1800 and 2200h and/or peak melatonin levels were observed during the night. Weight and spirometry were measured at the start and end of the therapy. Urinary biomarkers of kidney toxicity (KIM1, NAG, NGAL, IL-18 and CysC) were assayed at the start and end of the course of tobramycin.ResultsEighteen children were recruited to the study. There were no differences in renal clearance between the morning and evening groups. The increase in urinary KIM-1 was greater in the evening dosage group compared to the morning group (mean difference, 0.73ng/mg; 95% CI, 0.14 to 1.32; p=0.018). There were no differences in the other urinary biomarkers. There was normal circadian rhythm in 7/11 participants (64%).ConclusionsRenal elimination of tobramycin was not affected by the time of day of administration. Urinary KIM-1 raises the possibility of greater nephrotoxicity with evening administration. Four children showed disturbed circadian rhythm and high melatonin levels (ClinicalTrials.gov NCT01207245)

The effect of scleral search coil lens wear on the eye

BACKGROUND/AIM Scleral search coils are used to measure eye movements. A recent abstract suggests that the coil can affect the eye by decreasing visual acuity, increasing intraocular pressure, and damaging the corneal and conjunctival surface. Such findings, if repeated in all subjects, would cast doubt on the credibility of the search coil as a reliable investigative technique. The aim of this study was to reassess the effect of the scleral search coil on visual function. METHODS Six volunteer subjects were selected to undergo coil wear and baseline measurements were taken of logMAR visual acuity, non-contact tonometry, keratometry, and slit lamp examination. Four drops of 0.4% benoxinate hydrochloride were instilled before insertion of the lens by an experienced clinician. The lens then remained on the eye for 30 minutes. Measurements of the four ocular health parameters were repeated after 15 and 30 minutes of lens wear. The lens was then removed and the health of the eye reassessed. RESULTS No obvious pattern of change was found in logMAR visual acuity, keratometry, or intraocular pressure. The lens did produce changes to the conjunctival and corneal surfaces, but this was not considered clinically significant. CONCLUSION Search coils do not appear to cause any significant effects on visual function. However, thorough prescreening of subjects and post-wear checks should be carried out on all coil wearers to ensure no adverse effects have been caused

Investigating genome wide DNA methylation in bronchial and lung fibroblasts from healthy individuals and individuals with COPD

Rationale: Lung fibroblasts are implicated in respiratory disease pathology including chronic obstructive pulmonary disease (COPD). Phenotypic differences between fibroblasts isolated from the bronchi versus the lung parenchyma have been described but no studies have compared the cell types on a genome wide scale. DNA methylation is a reversible modification of the DNA structure with the ability to affect cell function via the alteration of gene expression. Here we compared genome wide DNA methylation profiles from bronchial and lung fibroblasts and assessed modification to these profiles in cells isolated from individuals with COPD. Methods: DNA was isolated from lung (LgF) and bronchial fibroblasts (BrF) at passage 4 and bisulphite treated. Site specific, quantitative genome wide methylation was determined using the Illumina 450K Infinium Methylation BeadChip array. Linear modelling and DMRcate functions identified differentially methylated sites and regions respectively between BrF and LgF and from cells isolated from healthy individuals versus those with COPD. Results: 3980 CpG (methylation) sites significantly differed, following Bonferroni correction, between BrF and LgF isolated from healthy individuals. These sites had a broad distribution of effect size, with 240 CpG sites displaying a difference in methylation of >50%. 78 of these sites were validated in a second cohort of matched BrF and LgF isolated from the same individuals. There was genomic proximity to these sites and DMRcate was used to refine the individual CpG sites to 5 regions of interest associated with 5 genes; HLX, TWIST1, CREB5, SKAP2 and PRDM16. Differences in methylation were less pronounced when comparing cells isolated from healthy individuals to those with COPD. In BrF 47 DMRcate regions were identified with a maximum difference in methylation of at least 20%. In LgF 3 DMRcate regions were identified with a maximum difference in methylation of at least 20%. Conclusions: DNA methylation profiles are significantly different between BrF and LgF but only small modifications are associated with COPD. Future work will focus on validating a methylation based marker of lung versus bronchial fibroblasts to differentiate cell types by validating our differential DNA methylation observations with gene/protein expression

Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds

Human airway smooth muscle (HASM) contraction plays a central role in regulating airway resistance in both healthy and asthmatic bronchioles. In vitro studies that investigate the intricate mechanisms that regulate this contractile process are predominantly conducted on tissue culture plastic, a rigid, 2D geometry, unlike the 3D microenvironment smooth muscle cells are exposed to in situ. It is increasingly apparent that cellular characteristics and responses are altered between cells cultured on 2D substrates compared with 3D topographies. Electrospinning is an attractive method to produce 3D topographies for cell culturing as the fibers produced have dimensions within the nanometer range, similar to cells' natural environment. We have developed an electrospun scaffold using the nondegradable, nontoxic, polymer polyethylene terephthalate (PET) composed of uniaxially orientated nanofibers and have evaluated this topography's effect on HASM cell adhesion, alignment, and morphology. The fibers orientation provided contact guidance enabling the formation of fully aligned sheets of smooth muscle. Moreover, smooth muscle cells cultured on the scaffold present an elongated cell phenotype with altered contractile protein levels and distribution. HASM cells cultured on this scaffold responded to the bronchoconstrictor bradykinin. The platform presented provides a novel in vitro model that promotes airway smooth muscle cell development toward a more in vivo-like phenotype while providing topological cues to ensure full cell alignment

Investigating genome wide dna methylation in airway and parenchymal fibroblasts from healthy individuals and individuals with copd

Rationale: Lung fibroblasts are implicated in respiratory disease pathology including chronic obstructive pulmonary disease (COPD). Phenotypic differences between fibroblasts isolated from the airway versus the parenchyma have been described but no studies have compared the cell types on a genome wide scale. DNA methylation is a reversible modification of the DNA structure with the ability to affect cell function via the alteration of gene expression. Here we compared genome wide DNA methylation profiles from airway and parenchymal fibroblasts and assessed modification to these profiles in cells isolated from individuals with COPD. Methods: DNA was isolated from parenchymal and airway fibroblasts at passage 4, and bisulphite treated. Site specific, quantitative genome wide methylation was determined using the Illumina 450K Infinium Methylation BeadChip array. Linear modelling and DMRcate functions identified differentially methylated sites and regions respectively between airway and parenchymal fibroblasts isolated from individuals with normal lung function versus those with COPD. Results: 3980 CpG (methylation) sites significantly differed after Bonferroni correction between airway and parenchymal fibroblasts isolated from healthy individuals. These sites had a broad distribution of effect size, with 240 CpG sites displaying a difference in methylation of >50%. 78 of these sites validated in a second cohort of 7 sets of paired airway and parenchymal fibroblasts isolated from the same individual. There was genomic proximity to these sites and DMRcate was used to refine the individual CpG sites to 5 regions of interest associated with 5 genes; HLX, TWIST1, CREB5, SKAP2 and PRDM16. Differences in methylation were less pronounced when comparing cells isolated from healthy individuals to those with COPD. In airway fibroblasts 47 DMRcate regions were identified with a maximum difference in methylation of at least 20%. In parenchymal fibroblasts 3 DMRcate regions were identified with a maximum difference in methylation of at least 20%. Conclusions: DNA methylation profiles are significantly different between airway and parenchymal fibroblasts but only small modifications are associated with COPD. Future work will focus on validating a methylation based markers of parenchymal versus airway fibroblasts and associating our differential observations with gene/protein expression

Investigating genome wide dna methylation in airway smooth muscle cells from asthmatic and non-asthmatic donors

Rationale: Genetic mechanisms fail to fully explain asthma pathogenesis and environmental factors are considered to play an important role. Environmental factors may lead to permanent changes in epigenetic patterns and contribute to asthma. Epigenetics is the study of heritable changes in gene expression that are not due to changes in DNA sequence. DNA methylation is a reversible modification of DNA structure in which a methyl group is added to cytosine residues. Parental smoking affects the methylation of buccal cell DNA from children and children with early onset wheeze have an altered blood DNA methylation profile to healthy individuals. No studies have compared DNA methylation profiles in the disease relevant cell type of airway smooth muscle (ASM) cells. Methods: DNA was isolated from ASM cells at passage 5 and bisulphite treated to convert epigenetic information into sequence-based information. Site specific, quantitative genome wide methylation was determined using the Illumina 450K Infinium Methylation BeadChip array. Hits were validated by Pyrosequencing. RNA was extracted simultaneously for mRNA expression analysis by real time PCR. Results: There were no independent CpG sites associated with asthmatic status of ASM cells following multiple test correction. Without correction over 13000 CpG sites showed a significant difference in methylation (linear modelling, p value >0.05) between asthmatic and non-asthmatic cells, and a biologically relevant difference in methylation of greater that 10% (β value >0.1 ). 10 of these sites were selected as top hits. 7 sites positively validated by pyrosequencing. They were associated with 7 different genes; LGALS3BP, ATP11A, ZNF696, KLF6, TBX1, RUNX3, and SPINT2. Expression of these genes was measured in ASM cells isolated from asthmatic and non-asthmatic donors. LGALS3BP expression was undetectable while ATP11A and ZNF696 displayed no difference in expression between cells from asthmatic and non-asthmatic donors. KLF6 and SPINT2 showed a trend towards increased expression in cells from asthmatic donors while RUNX3 and TBX1 showed a trend towards decreased expression. Conclusions: Differences in CpG methylation exist between ASM isolated from asthmatic and non-asthmatic donors. Future work will focus on identifying differentially methylated regions of DNA and further defining the association to gene and protein expression

Caching and Interpolated Likelihoods: Accelerating Cosmological Monte Carlo Markov Chains

We describe a novel approach to accelerating Monte Carlo Markov Chains. Our focus is cosmological parameter estimation, but the algorithm is applicable to any problem for which the likelihood surface is a smooth function of the free parameters and computationally expensive to evaluate. We generate a high-order interpolating polynomial for the log-likelihood using the first points gathered by the Markov chains as a training set. This polynomial then accurately computes the majority of the likelihoods needed in the latter parts of the chains. We implement a simple version of this algorithm as a patch (InterpMC) to CosmoMC and show that it accelerates parameter estimatation by a factor of between two and four for well-converged chains. The current code is primarily intended as a "proof of concept", and we argue that there is considerable room for further performance gains. Unlike other approaches to accelerating parameter fits, we make no use of precomputed training sets or special choices of variables, and InterpMC is almost entirely transparent to the user.Comment: v2 Trivial Latex change. Source code: http://easther.physics.yale.edu/interpmc.htm