Cluster-specific associations between the gut microbiota and behavioral outcomes in preschool-aged children.

BACKGROUND: The gut microbiota is recognized as a regulator of brain development and behavioral outcomes during childhood. Nonetheless, associations between the gut microbiota and behavior are often inconsistent among studies in humans, perhaps because many host-microbe relationships vary widely between individuals. This study aims to stratify children based on their gut microbiota composition (i.e., clusters) and to identify novel gut microbiome cluster-specific associations between the stool metabolomic pathways and child behavioral outcomes. METHODS: Stool samples were collected from a community sample of 248 typically developing children (3-5 years). The gut microbiota was analyzed using 16S sequencing while LC-MS/MS was used for untargeted metabolomics. Parent-reported behavioral outcomes (i.e., Adaptive Skills, Internalizing, Externalizing, Behavioral Symptoms, Developmental Social Disorders) were assessed using the Behavior Assessment System for Children (BASC-2). Children were grouped based on their gut microbiota composition using the Dirichlet multinomial method, after which differences in the metabolome and behavioral outcomes were investigated. RESULTS: Four different gut microbiota clusters were identified, where the cluster enriched in both Bacteroides and Bifidobacterium (Ba2) had the most distinct stool metabolome. The cluster characterized by high Bifidobacterium abundance (Bif), as well as cluster Ba2, were associated with lower Adaptive Skill scores and its subcomponent Social Skills. Cluster Ba2 also had significantly lower stool histidine to urocanate turnover, which in turn was associated with lower Social Skill scores in a cluster-dependent manner. Finally, cluster Ba2 had increased levels of compounds involved in Galactose metabolism (i.e., stachyose, raffinose, alpha-D-glucose), where alpha-D-glucose was associated with the Adaptive Skill subcomponent Daily Living scores (i.e., ability to perform basic everyday tasks) in a cluster-dependent manner. CONCLUSIONS: These data show novel associations between the gut microbiota, its metabolites, and behavioral outcomes in typically developing preschool-aged children. Our results support the concept that cluster-based groupings could be used to develop more personalized interventions to support child behavioral outcomes. Video Abstract.

Exploring associations between the gut microbiota and full-scale intelligence in preschool children.

The relationship between the gut microbiota and neurocognitive outcomes is becoming increasingly recognized; however, findings in humans are inconsistent. In addition, few studies have investigated the gut microbial metabolites that may mediate this relationship. The objective of this study was to investigate associations between full-scale intelligence (FSIQ) and the composition of the gut microbiota and metabolome in preschool children. Stool samples were collected from a community sample of 245 typically developing children (3-5 years) from the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort. The faecal microbiome was assessed using 16S rRNA sequencing and the metabolome using LC-MS/MS. FSIQ and scores on the Verbal Comprehension, Visual Spatial, Working Memory indices of the Wechsler Preschool and Primary Scale of Intelligence-IV were used to assess neurocognition. Associations between the gut microbiota and FSIQ were determined using Pearson and Spearman correlations, which were corrected for multiple testing and relevant covariates. Verbal Comprehension correlated negatively with both Shannon alpha diversity (r = -0.14, p = 0.032) and the caffeine-derived metabolite paraxanthine (r = -0.22, p < 0.001). No other significant correlations were observed. Overall, the weak to modest correlations between Verbal Comprehension with alpha diversity and paraxanthine provide limited evidence of an association between the gut microbiota and neurocognitive outcomes in typically developing preschool children.

Next-generation sequencing approach to investigate genome variability of Parapoxvirus in Canadian muskoxen (Ovibos moschatus).

In 2016, the first orf virus, a double-stranded DNA (dsDNA) virus of the genus parapoxvirus, from a muskox was isolated on Victoria Island, Nunavut (NU), Canada. We used deep sequencing on DNA extracted from orf virus-positive tissues from wild muskoxen from locations on Victoria Island and the adjacent mainland. Orf virus sequence reads derived from four samples were nearly identical. The consensus sequences generated from pooled reads of MxOV comprises of a large contiguous sequence (contig) of 131,759 bp and a smaller right terminal contig of 3552 bp, containing all coding sequences identified as Parapoxvirus. Individual gene comparisons reveal that MxOV shares genetic characteristics with reference strains from both sheep and goat origin. Recombination analysis using Bootscan, MAXCHI, GENECONV, CHIMAERA, SISCAN, and RDP algorithms within the RDP4 software predicted recombination events in two virulence factors, and a large 3000 bp segment of the MxOV genome. Partial B2L nucleotide sequences from strains around the world and other North American isolates were compared to MxOV using MUSCLE alignments and RAxML phylogenetic trees. MxOV was identical to our previously characterized isolate, and shared similarity with orf virus isolated from sheep and goats. The phylogenetic grouping of partial B2L nucleotide sequences did not follow the sample geographic distribution. More full genomes of orf virus, or at least full B2L gene squences, in wildlife are needed especially in North America to better understand the epidemiology of the disease in muskoxen.

Genomic signatures of selection associated with benzimidazole drug treatments in Haemonchus contortus field populations.

Genome-wide methods offer a powerful approach to detect signatures of drug selection. However, limited availability of suitable reference genomes and the difficulty of obtaining field populations with well-defined, distinct drug treatment histories mean there is little information on the signatures of selection in parasitic nematodes and on how best to detect them. This study addresses these knowledge gaps by using field populations of Haemonchus contortus with well-defined benzimidazole treatment histories, leveraging a recently completed chromosomal-scale reference genome assembly. We generated a panel of 49,393 genomic markers to genotype 20 individual adult worms from each of four H. contortus populations: two from closed sheep flocks with an approximate 20 year history of frequent benzimidazole treatment, and two populations with a history of little or no treatment. Sampling occurred in the same geographical region to limit genetic differentiation and maximise the detection sensitivity. A clear signature of selection was detected on chromosome I, centred on the isotype-1 ß-tubulin gene. Two additional, but weaker, signatures of selection were detected; one near the middle of chromosome I spanning 3.75 Mbp and 259 annotated genes, and one on chromosome II spanning a region of 3.3 Mbp and 206 annotated genes, including the isotype-2 ß-tubulin locus. We also assessed how sensitivity was impacted by sequencing depth, worm number, and pooled versus individual worm sequence data. This study provides the first known direct genome-wide evidence for any parasitic nematode, that the isotype-1 ß-tubulin gene is quantitatively the single most important benzimidazole resistance locus. It also identified two additional genomic regions that likely contain benzimidazole resistance loci of secondary importance. This study provides an experimental framework to maximise the power of genome-wide approaches to detect signatures of selection driven by anthelmintic drug treatments in field populations of parasitic nematodes.

Associations Between the Gut Microbiota and Internalizing Behaviors in Preschool Children.

OBJECTIVE: Emerging evidence points toward a connection between mental health and the gut microbiota and its metabolites (e.g., short-chain fatty acids). It is unknown whether the gut microbiota is associated with the development of mental health problems (e.g., internalizing or externalizing behaviors) in preschool children. The objective of this study was to evaluate associations between the gut microbiota and internalizing and externalizing behaviors in preschool-aged children. METHODS: A community sample of 248 typically developing children (3-5 years of age) provided a stool sample for gut microbiota and SCFA analysis. Parents reported child internalizing and externalizing behaviors using the Child Behavior Checklist. Associations between child behaviors and gut microbiota measures were analyzed using Spearman correlations followed by an adjustment for multiple testing, with subanalysis conducted in children clinically "at risk" for behavioral problems compared with those who were not. RESULTS: There was a correlation between Shannon alpha diversity with internalizing behaviors (rs = -0.134, p = .035) and its subscale somatic complaints (rs = -0.144, p = .023). In addition, children clinically "at risk" for internalizing problems had decreased alpha diversity (U = 551, p = .017). Internalizing behaviors correlated with valerate and isobutyrate (rs = -0.147, p = .021; rs = -0.140, p = .028, respectively). Furthermore the somatic complaints subscale additionally correlated with acetate and butyrate (rs = -0.219, p = .001; rs = -0.241, p < .001, respectively). These findings were also present in children "at risk" for internalizing problems (U = 569, p = .026; U = 571, p = .028) and somatic complaints (U = 164, p = .004; U = 145, p = .001). CONCLUSIONS: These analyses reveal novel associations between internalizing behaviors and the gut microbiota in preschool children. Furthermore, a relationship between somatic complaints and acetate and butyrate was identified, indicating that interventions that increase SCFA production warrant future investigation.

Characterization of the ß-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance.

BACKGROUND: The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding ß-tubulin proteins, the principal targets of BZ drugs. METHODOLOGY AND PRINCIPAL FINDINGS: First, the ß-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different ß-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. SIGNIFICANCE: This study demonstrated the presence of at least seven ß-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the ß-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of ß-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

Characterization of microglial transcriptomes in the brain and spinal cord of mice in early and late experimental autoimmune encephalomyelitis using a RiboTag strategy.

Microglia play an important role in the pathogenesis of multiple sclerosis and the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). To more fully understand the role of microglia in EAE we characterized microglial transcriptomes before the onset of motor symptoms (pre-onset) and during symptomatic EAE. We compared the transcriptome in brain, where behavioral changes are initiated, and spinal cord, where damage is revealed as motor and sensory deficits. We used a RiboTag strategy to characterize ribosome-bound mRNA only in microglia without incurring possible transcriptional changes after cell isolation. Brain and spinal cord samples clustered separately at both stages of EAE, indicating regional heterogeneity. Differences in gene expression were observed in the brain and spinal cord of pre-onset and symptomatic animals with most profound effects in the spinal cord of symptomatic animals. Canonical pathway analysis revealed changes in neuroinflammatory pathways, immune functions and enhanced cell division in both pre-onset and symptomatic brain and spinal cord. We also observed a continuum of many pathways at pre-onset stage that continue into the symptomatic stage of EAE. Our results provide additional evidence of regional and temporal heterogeneity in microglial gene expression patterns that may help in understanding mechanisms underlying various symptomology in MS.

Crohn's Disease Pathobiont Adherent-Invasive E coli Disrupts Epithelial Mitochondrial Networks With Implications for Gut Permeability.

BACKGROUND & AIMS: Adherent-invasive Escherichia coli are implicated in inflammatory bowel disease, and mitochondrial dysfunction has been observed in biopsy specimens from patients with inflammatory bowel disease. As a novel aspect of adherent-invasive E coli-epithelial interaction, we hypothesized that E coli (strain LF82) would elicit substantial disruption of epithelial mitochondrial form and function. METHODS: Monolayers of human colon-derived epithelial cell lines were exposed to E coli-LF82 or commensal E coli and RNA sequence analysis, mitochondrial function (adenosine triphosphate synthesis) and dynamics (mitochondrial network imaging, immunoblotting for fission and fusion proteins), and epithelial permeability (transepithelial resistance, flux of fluorescein isothiocyanate-dextran and bacteria) were assessed. RESULTS: E coli-LF82 significantly affected epithelial expression of ∼8600 genes, many relating to mitochondrial function. E coli-LF82-infected epithelia showed swollen mitochondria, reduced mitochondrial membrane potential and adenosine triphosphate, and fragmentation of the mitochondrial network: events not observed with dead E coli-LF82, medium from bacterial cultures, or control E coli. Treatment with Mitochondrial Division Inhibitor 1 (Mdivi1, inhibits dynamin-related peptide 1, guanosine triphosphatase principally responsible for mitochondrial fission) or P110 (prevents dynamin-related peptide 1 binding to mitochondrial fission 1 protein) partially reduced E coli-LF82-induced mitochondrial fragmentation in the short term. E coli-LF82-infected epithelia showed loss of the long isoform of optic atrophy factor 1, which mediates mitochondrial fusion. Mitochondrial Division Inhibitor 1 reduced the magnitude of E coli-LF82-induced increased transepithelial flux of fluorescein isothiocyanate dextran. By 8 hours after infection, increased cytosolic cytochrome C and DNA fragmentation were apparent without evidence of caspase-3 or apoptosis inducing factor activation. CONCLUSIONS: Epithelial mitochondrial fragmentation caused by E coli-LF82 could be targeted to maintain cellular homeostasis and mitigate infection-induced loss of epithelial barrier function. Data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO series accession numbers GSE154121 and GSE154122 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154121).

A repeatable and quantitative DNA metabarcoding assay to characterize mixed strongyle infections in horses.

Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae from fecal samples from an experimental herd in Kentucky, USA and two feral horse populations from Sable Island and Alberta, Canada. In addition to reporting the detection of 33 different species with high confidence, we illustrate the assay's repeatability by comparing results generated from aliquots from the same fecal samples and from individual horses sampled repeatedly over multiple days or months. We also validate the quantitative potential of the assay by demonstrating that the proportion of amplicon reads assigned to different species scales linearly with the number of larvae present. This new tool significantly improves equine strongyle diagnostics, presenting opportunities for research on species-specific anthelmintic resistance and the causes and consequences of variation in mixed infections.

A database for ITS2 sequences from nematodes.

BACKGROUND: Marker gene surveys have a wide variety of applications in species identification, population genetics, and molecular epidemiology. As these methods expand to new types of organisms and additional markers beyond 16S and 18S rRNA genes, comprehensive databases are a critical requirement for proper analysis of these data. RESULTS: Here we present an ITS2 rDNA database for marker gene surveys of both free-living and parasitic nematode populations and the software used to build the database. This is currently the most complete and up-to-date ITS2 database for nematodes and is able to reproduce previous analysis that used a smaller database. CONCLUSIONS: This database is an important resource for researchers working on nematodes and also provides a tool to create ITS2 databases for any given taxonomy.

Human interleukin-4-treated regulatory macrophages promote epithelial wound healing and reduce colitis in a mouse model.

Murine alternatively activated macrophages can exert anti-inflammatory effects. We sought to determine if IL-4-treated human macrophages [i.e., hM(IL4)] would promote epithelial wound repair and can serve as a cell transfer treatment for inflammatory bowel disease (IBD). Blood monocytes from healthy volunteers and patients with active and inactive IBD were converted to hM(IL4)s. IL-4 treatment of blood-derived macrophages from healthy volunteers and patients with inactive IBD resulted in a characteristic CD206+CCL18+CD14low/- phenotype (RNA-seq revealed IL-4 affected expression of 996 genes). Conditioned media from freshly generated or cryopreserved hM(IL4)s promoted epithelial wound healing in part by TGF, and reduced cytokine-driven loss of epithelial barrier function in vitro. Systemic delivery of hM(IL4) to dinitrobenzene sulphonic acid (DNBS)-treated Rag1-/- mice significantly reduced disease. These findings from in vitro and in vivo analyses provide proof-of-concept support for the development of autologous M(IL4) transfer as a cellular immunotherapy for IBD.

Cystic Fibrosis Patients Infected With Epidemic Pseudomonas aeruginosa Strains Have Unique Microbial Communities.

Pseudomonas aeruginosa is the archetypal cystic fibrosis (CF) pathogen. However, the clinical course experienced by infected individuals varies markedly. Understanding these differences is imperative if further improvements in outcomes are to be achieved. Multiple studies have found that patients infected with epidemic P. aeruginosa (ePA) strains may have a worse clinical prognosis than those infected with unique, non-clonal strains. Additionally, the traditionally uncultured CF lung bacterial community (i.e., CF microbiome) may further influence the outcome. We sought to identify if these two important variables, not identified through routine culture, associate and together may contribute to disease pathogenesis. Patients were classified as being infected with Prairie Epidemic ePA (PES) or a non-clonal strain, unique PA strains (uPA), through a retrospective assessment of a comprehensive strain biobank using a combination of PFGE and PES-specific PCR. Patients were matched to age, sex, time-period controls and sputum samples from equivalent time periods were identified from a sputum biobank. Bacterial 16S rRNA gene profiling and Pseudomonas qPCR was used to characterize the respiratory microbiome. We identified 31 patients infected with PES and matched them with uPA controls. Patients infected with PES at baseline have lower microbial diversity (P = 0.02) and higher P. aeruginosa relative abundance (P < 0.005). Microbial community structure was found to cluster by PA strain type, although it was not the main determinant of community structure as additional factors were also found to be drivers of CF community structure. Communities from PES infected individuals were enriched with Pseudomonas, Streptococcus and Prevotella OTUs. The disproportionate disease experienced by ePA infected CF patients may be mediated through a combination of pathogen-pathogen factors as opposed to strictly enhanced virulence of infecting P. aeruginosa strains.

Th1-Th2 Cross-Regulation Controls Early Leishmania Infection in the Skin by Modulating the Size of the Permissive Monocytic Host Cell Reservoir.

The impact of T helper (Th) 1 versus Th2 immunity on intracellular infections is attributed to classical versus alternative activation of macrophages leading to resistance or susceptibility. However, observations in multiple infectious settings demonstrate deficiencies in mediators of Th1-Th2 immunity, which have paradoxical or no impact. We report that prior to influencing activation, Th1/Th2 immunity first controls the size of the permissive host cell reservoir. During early Leishmania infection of the skin, IFN-γ- or STAT6-mediated changes in phagocyte activation were counteracted by changes in IFN-γ-mediated recruitment of permissive CCR2+ monocytes. Monocytes were required for early parasite expansion and acquired an alternatively activated phenotype despite the Th1 dermal environment required for their recruitment. Surprisingly, STAT6 did not enhance intracellular parasite proliferation, but rather modulated the size and permissiveness of the monocytic host cell reservoir via regulation of IFN-γ and IL-10. These observations expand our understanding of the Th1-Th2 paradigm during infection.

Benchmarking hybrid assemblies of Giardia and prediction of widespread intra-isolate structural variation.

BACKGROUND: Currently available short read genome assemblies of the tetraploid protozoan parasite Giardia intestinalis are highly fragmented, highlighting the need for improved genome assemblies at a reasonable cost. Long nanopore reads are well suited to resolve repetitive genomic regions resulting in better quality assemblies of eukaryotic genomes. Subsequent addition of highly accurate short reads to long-read assemblies further improves assembly quality. Using this hybrid approach, we assembled genomes for three Giardia isolates, two with published assemblies and one novel, to evaluate the improvement in genome quality gained from long reads. We then used the long reads to predict structural variants to examine this previously unexplored source of genetic variation in Giardia. METHODS: With MinION reads for each isolate, we assembled genomes using several assemblers specializing in long reads. Assembly metrics, gene finding, and whole genome alignments to the reference genomes enabled direct comparison to evaluate the performance of the nanopore reads. Further improvements from adding Illumina reads to the long-read assemblies were evaluated using gene finding. Structural variants were predicted from alignments of the long reads to the best hybrid genome for each isolate and enrichment of key genes was analyzed using random genome sampling and calculation of percentiles to find thresholds of significance. RESULTS: Our hybrid assembly method generated reference quality genomes for each isolate. Consistent with previous findings based on SNPs, examination of heterozygosity using the structural variants found that Giardia BGS was considerably more heterozygous than the other isolates that are from Assemblage A. Further, each isolate was shown to contain structural variant regions enriched for variant-specific surface proteins, a key class of virulence factor in Giardia. CONCLUSIONS: The ability to generate reference quality genomes from a single MinION run and a multiplexed MiSeq run enables future large-scale comparative genomic studies within the genus Giardia. Further, prediction of structural variants from long reads allows for more in-depth analyses of major sources of genetic variation within and between Giardia isolates that could have effects on both pathogenicity and host range.

Complex patterns of cell growth in the placenta in normal pregnancy and as adaptations to maternal diet restriction.

The major milestones in mouse placental development are well described, but our understanding is limited to how the placenta can adapt to damage or changes in the environment. By using stereology and expression of cell cycle markers, we found that the placenta grows under normal conditions not just by hyperplasia of trophoblast cells but also through extensive polyploidy and cell hypertrophy. In response to feeding a low protein diet to mothers prior to and during pregnancy, to mimic chronic malnutrition, we found that this normal program was altered and that it was influenced by the sex of the conceptus. Male fetuses showed intrauterine growth restriction (IUGR) by embryonic day (E) 18.5, just before term, whereas female fetuses showed IUGR as early as E16.5. This difference was correlated with differences in the size of the labyrinth layer of the placenta, the site of nutrient and gas exchange. Functional changes were implied based on up-regulation of nutrient transporter genes. The junctional zone was also affected, with a reduction in both glycogen trophoblast and spongiotrophoblast cells. These changes were associated with increased expression of Phlda2 and reduced expression of Egfr. Polyploidy, which results from endoreduplication, is a normal feature of trophoblast giant cells (TGC) but also spongiotrophoblast cells. Ploidy was increased in sinusoidal-TGCs and spongiotrophoblast cells, but not parietal-TGCs, in low protein placentas. These results indicate that the placenta undergoes a range of changes in development and function in response to poor maternal diet, many of which we interpret are aimed at mitigating the impacts on fetal and maternal health.

foxc1 is required for embryonic head vascular smooth muscle differentiation in zebrafish.

Vascular smooth muscle of the head derives from neural crest, but developmental mechanisms and early transcriptional drivers of the vSMC lineage are not well characterized. We find that in early development, the transcription factor foxc1b is expressed in mesenchymal cells that associate with the vascular endothelium. Using timelapse imaging, we observe that foxc1b expressing mesenchymal cells differentiate into acta2 expressing vascular mural cells. We show that in zebrafish, while foxc1b is co-expressed in acta2 positive smooth muscle cells that associate with large diameter vessels, it is not co-expressed in capillaries where pdgfrß positive pericytes are located. In addition to being an early marker of the lineage, foxc1 is essential for vSMC differentiation; we find that foxc1 loss of function mutants have defective vSMC differentiation and that early genetic ablation of foxc1b or acta2 expressing populations blocks vSMC differentiation. Furthermore, foxc1 is expressed upstream of acta2 and is required for acta2 expression in vSMCs. Using RNA-Seq we determine an enriched intersectional gene expression profile using dual expression of foxc1b and acta2 to identify novel vSMC markers. Taken together, our data suggests that foxc1 is a marker of vSMCs and plays a critical functional role in promoting their differentiation.

The effects of cycled inhaled aztreonam on the cystic fibrosis (CF) lung microbiome.

BACKGROUND: To improve clinical outcomes, cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa infections are prescribed inhaled anti-pseudomonal antibiotics. Although, a diverse microbial community exists within CF airways, little is known about how the CF microbiota influences patient outcomes. We hypothesized that organisms within the CF microbiota are affected by inhaled-antibiotics and baseline microbiome may be used to predict therapeutic response. METHODS: Adults with chronic P. aeruginosa infection from four clinics were observed during a single 28-day on/off inhaled-aztreonam cycle. Patients performed serial sputum collection, CF-respiratory infection symptom scores (CRISS), and spirometry. Patients achieving a decrease of ≥2 CRISS by day 28 were categorized as subjective responders (SR). The airway microbiome was defined by Illumina MiSeq analysis of the 16S rRNA gene. RESULTS: Thirty-seven patients (median 37.4 years and FEV1 44% predicted) were enrolled. No significant cohort-wide changes in the microbiome were observed between on/off AZLI cycles in either alpha- or beta-diversity metrics. However, at an individual level shifts were apparent. Twenty-one patients (57%) were SR and fourteen patients did not subjectively respond. While alpha-diversity metrics did not associate with response, patients who did not subjectively respond had a higher abundance of Staphylococcus and Streptococcus, and lower abundance of Haemophilus. CONCLUSIONS: The CF microbiome is relatively resilient to AZLI perturbations. However, associated changes were observed at the individual patient level. The relative abundance of key "off-target" organisms associated with subjective improvements suggesting that the microbiome may be used as a tool to predict patient response - potentially improving outcomes.

Host genetics and diet composition interact to modulate gut microbiota and predisposition to metabolic syndrome in spontaneously hypertensive stroke-prone rats.

Metabolic syndrome encompasses obesity, glucose intolerance, hypertension, and dyslipidemia; however, the interactions between diet and host physiology that predispose to metabolic syndrome are incompletely understood. Here, we explored the effects of a high-fat diet (HFD) on energy balance, gut microbiota, and risk factors of metabolic syndrome in spontaneously hypertensive stroke-prone (SHRSP) and Wistar-Kyoto (WKY) rats. We found that the SHRSP rats were hypertensive, hyperphagic, less sensitive to hypophagic effects of exendin-4, and expended more energy with diminished sensitivity to sympathetic blockade compared to WKY rats. Notably, key thermogenic markers in brown and retroperitoneal adipose tissues and skeletal muscle were up-regulated in SHRSP than WKY rats. Although HFD promoted weight gain, adiposity, glucose intolerance, hypertriglyceridemia, hepatic lipidosis, and hyperleptinemia in both SHRSP and WKY rats, the SHRSP rats weighed less but had comparable percent adiposity to WKY rats, which supports the use of HFD-fed SHRSP rats as a unique model for studying the metabolically obese normal weight (MONW) phenotype in humans. Despite distinct strain differences in gut microbiota composition, diet had a preponderant impact on gut flora with some of the taxa being strongly associated with key metabolic parameters. Together, we provide evidence that interactions between host genetics and diet modulate gut microbiota and predispose SHRSP rats to develop metabolic syndrome.-Singh, A., Zapata, R. C., Pezeshki, A., Workentine, M. L., Chelikani, P. K. Host genetics and diet composition interact to modulate gut microbiota and predisposition to metabolic syndrome in spontaneously hypertensive stroke-prone rats.

Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Macrophages play central roles in immunity as early effectors and modulating adaptive immune reponses; we implicated macrophages in the anticolitic effect of infection with the tapeworm Hymenolepis diminuta. Here, gene arrays revealed that H. diminuta antigen (HdAg) evoked a program in murine macrophages distinct from that elicited by IL-4. Further, HdAg suppressed LPS-evoked release of TNF-α and IL-1ß from macrophages via autocrine IL-10 signaling. In assessing the ability of macrophages treated in vitro with an extract of H. diminuta [M(HdAg)] to affect disease, intravenous, but not peritoneal, injection of M(HdAg) protected wild-type but not RAG1-/- mice from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Administration of splenic CD4+ T cells from in vitro cocultures with M(HdAg), but not those cocultured with M(IL-4) cells, inhibited DNBS-induced colitis; fractionation of the T-cell population indicated that the CD4+CD25+ T cells from cocultures with M(HdAg) drove the suppression of DNBS-induced colitis. Use of IL-4-/- or IL-10-/- CD4+ T cells revealed that neither cytokine alone from the donor cells was essential for the anticolitic effect. These data illustrate that HdAg evokes a unique regulatory program in macrophages, identifies HdAg-evoked IL-10 suppression of macrophage activation, and reveals the ability of HdAg-treated macrophages to educate ( i.e., condition) and mobilize CD4+CD25+ T cells, which could be deployed to treat colonic inflammation.-Reyes, J. L., Lopes, F., Leung, G., Jayme, T. S., Matisz, C. E., Shute, A., Burkhard, R., Carneiro, M., Workentine, M. L., Wang, A., Petri, B., Beck, P. L., Geuking, M. B., McKay, D. M., Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle.

The objective was to study effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of feedlot cattle. Two groups of 30 Angus-cross heifers were studied from weaning to 28 d after arrival at a feedlot. For each group, half the heifers were either transported directly to a feedlot after weaning (RANC) or transported to and co-mingled at an auction market for 24 h before being placed in a feedlot (AUCT). Deep nasal swabs (DNS) and trans-tracheal aspirates (TTA) were collected at weaning (d0) and at on-arrival processing at the feedlot (d2). At 7 (d9) and 28 d (d30) after arrival, DNS were repeated. The DNA was extracted from DNS and TTA and the V4 region of the 16S rRNA gene sequenced (MiSeq). Alpha diversity analysis did not reveal differences between AUCT and RANC. However, bacterial diversity decreased over time in the nasopharynx, especially at d9. Although beta-diversity was not different between AUCT and RANC, interval after arrival and feedlot where heifers were placed affected composition of the nasopharyngeal bacterial communities. In both groups, a large increase in Mycoplasma was observed after arrival; in one group, Mycoplasma bovis was dominant at d9 and remained dominant until d30. However, in the other group, Mycoplasma dispar dominated at d9 and was replaced by Moraxella at d30. We concluded that transportation to and co-mingling at an auction market for 24 h did not significantly influence diversity or composition of nasopharyngeal or tracheal bacterial communities.