Temporal and long-term gut microbiota variation in allergic disease: A prospective study from infancy to school age.

BACKGROUND: Compositional changes in the early-life gut microbiota have been implicated in IgE-associated allergic diseases, but there is lack of longitudinal studies. We examined gut microbiota development from infancy to school age in relation to onset of IgE-associated allergic diseases. At 8 years of age, we also examined the relationship between gut microbiota and T-cell regulation, estimated as responses to polyclonal T-cell activation. METHODS: Stool samples were collected from 93 children at 4, 6, 13 months, and 8 years of age. The gut microbiota was profiled using 16S rRNA gene sequencing. Peripheral blood was drawn from all children, and mononuclear cells were polyclonally activated. Levels of IL-10 and FOXP3 mRNA copies were determined using real-time quantitative reverse transcriptase-PCR. RESULTS: At 8 years of age, 21 children were diagnosed with IgE-associated allergic disease and 90% displayed allergic comorbidity. Seventy-two children were nonallergic and nonsensitized. Statistical tests with multiple testing corrections demonstrated temporal underrepresentation of Ruminococcus and consistent underrepresentation of Bacteroides, Prevotella, and Coprococcus in allergic compared to nonallergic children from infancy to school age. The gut microbiota of the allergic 8-year-olds was enriched in Bifidobacterium and depleted of Lactobacillus, Enterococcus, and Lachnospira. In allergic 8-year-olds, Faecalibacterium correlated with IL-10 mRNA levels (rs  = 0.49, Padj  = 0.02) with the same trend for FOXP3 (rs  = 0.39, Padj  = 0.08). CONCLUSIONS: We identified both temporal and long-term variation in the differential abundance of specific bacterial genera in children developing IgE-associated allergic disease. Improved dietary interventions aiming at expanding immune-modulatory taxa could be studied for prevention of allergic disease.

A new concept and a comprehensive evaluation of SYSMEX UF-1000i flow cytometer to identify culture-negative urine specimens in patients with UTI.

Urinary tract infections (UTIs) are among the most common bacterial infections in men and urine culture is gold standard for diagnosis. Considering the high prevalence of culture-negative specimens, any method that identifies such specimens is of interest. The aim was to evaluate a new screening concept for flow cytometry analysis (FCA). The outcomes were evaluated against urine culture, uropathogen species and three conventional screening methods. A prospective, consecutive study examined 1,312 urine specimens, collected during January and February 2012. The specimens were analyzed using the Sysmex UF1000i FCA. Based on the FCA data culture negative specimens were identified in a new model by use of linear discriminant analysis (FCA-LDA). In total 1,312 patients were included. In- and outpatients represented 19.6% and 79.4%, respectively; 68.3% of the specimens originated from women. Of the 610 culture-positive specimens, Escherichia coli represented 64%, enterococci 8% and Klebsiella spp. 7%. Screening with FCA-LDA at 95% sensitivity identified 42% (552/1312) as culture negative specimens when UTI was defined according to European guidelines. The proposed screening method was either superior or similar in comparison to the three conventional screening methods. In conclusion, the proposed/suggested and new FCA-LDA screening method was superior or similar to three conventional screening methods. We recommend the proposed screening method to be used in clinic to exclude culture negative specimens, to reduce workload, costs and the turnaround time. In addition, the FCA data may add information that enhance handling and support diagnosis of patients with suspected UTI pending urine culture [corrected].

High-risk regions and outbreak modelling of tularemia in humans.

Sweden reports large and variable numbers of human tularemia cases, but the high-risk regions are anecdotally defined and factors explaining annual variations are poorly understood. Here, high-risk regions were identified by spatial cluster analysis on disease surveillance data for 1984-2012. Negative binomial regression with five previously validated predictors (including predicted mosquito abundance and predictors based on local weather data) was used to model the annual number of tularemia cases within the high-risk regions. Seven high-risk regions were identified with annual incidences of 3·8-44 cases/100 000 inhabitants, accounting for 56·4% of the tularemia cases but only 9·3% of Sweden's population. For all high-risk regions, most cases occurred between July and September. The regression models explained the annual variation of tularemia cases within most high-risk regions and discriminated between years with and without outbreaks. In conclusion, tularemia in Sweden is concentrated in a few high-risk regions and shows high annual and seasonal variations. We present reproducible methods for identifying tularemia high-risk regions and modelling tularemia cases within these regions. The results may help health authorities to target populations at risk and lay the foundation for developing an early warning system for outbreaks.

Gut microbiome and innate immune response patterns in IgE-associated eczema.

BACKGROUND: Gut microbiome patterns have been associated with predisposition to eczema potentially through modulation of innate immune signalling. OBJECTIVE: We examined gut microbiome development in the first year of life in relation to innate immune responses and onset of IgE-associated eczema over the first 2.5 years in predisposed children due to maternal atopy [www.anzctr.org.au, trial ID ACTRN12606000280505]. METHODS: Microbial composition and diversity were analysed with barcoded 16S rRNA 454 pyrosequencing in stool samples in pregnancy and at ages 1 week, 1 month and 12 months in infants (n = 10) who developed IgE-associated eczema and infants who remained free of any allergic symptoms at 2.5 years of age (n = 10). Microbiome data at 1 week and 1 month were analysed in relation to previously assessed immune responses to TLR 2 and 4 ligands at 6 months of age. RESULTS: The relative abundance of Gram-positive Ruminococcaceae was lower at 1 week of age in infants developing IgE-associated eczema, compared with controls (P = 0.0047). At that age, the relative abundance of Ruminococcus was inversely associated with TLR2 induced IL-6 (-0.567, P = 0.042) and TNF-α (-0.597, P = 0.032); there was also an inverse association between the abundance of Proteobacteria (comprising Gram-negative taxa) and TLR4-induced TNF-α (rs = -0.629, P = 0.024). This relationship persisted at 1 month, with inverse associations between the relative abundance of Enterobacteriaceae (within the Proteobacteria phylum) and TLR4-induced TNF-α (rs = -0.697, P = 0.038) and Enterobacteriaceae and IL-6 (rs = -0.709, P = 0.035). Mothers whose infants developed IgE-associated eczema had lower α-diversity of Bacteroidetes (P = 0.04) although this was not seen later in their infants. At 1 year, α-diversity of Actinobacteria was lower in infants with IgE-associated eczema compared with controls (P = 0.002). CONCLUSION AND CLINICAL RELEVANCE: Our findings suggest that reduced relative abundance of potentially immunomodulatory gut bacteria is associated with exaggerated inflammatory cytokine responses to TLR-ligands and subsequent development of IgE-associated eczema.

What happens to food choices when a gluten-free diet is required? A prospective longitudinal population-based study among Swedish adolescent with coeliac disease and their peers.

A dietary survey was performed during a large screening study in Sweden among 13-year-old adolescents. The aim was to study how the intake of food groups was affected by a screening-detected diagnosis of coeliac disease (CD) and its gluten-free (GF) treatment. Food intake was reported using a FFQ, and intake reported by the adolescents who were diagnosed with CD was compared with the intake of two same-aged referent groups: (i) adolescents diagnosed with CD prior to screening; and (ii) adolescents without CD. The food intake groups were measured at baseline before the screening-detected cases were aware of their CD, and 12-18 months later. The results showed that food intakes were affected by screen-detected CD and its dietary treatment. Many flour-based foods were reduced such as pizza, fish fingers and pastries. The results also indicated that bread intake was lower before the screened diagnosis compared with the other studied groups, but increased afterwards. Specially manufactured GF products (for example, pasta and bread) were frequently used in the screened CD group after changing to a GF diet. The present results suggest that changing to a GF diet reduces the intake of some popular foods, and the ingredients on the plate are altered, but this do not necessarily include a change of food groups. The availability of manufactured GF replacement products makes it possible for adolescents to keep many of their old food habits when diagnosed with CD in Sweden.

miRNA analysis of formalin-fixed squamous cell carcinomas of the tongue is affected by age of the samples.

Global miRNA expression arrays were used for analysis of 836 miRNAs in formalin-fixed paraffin-embedded samples from 21 tongue cancer patients and 8 controls. Samples had been stored for one to eleven years. Results separated tumour samples from controls, however, the largest variation was correlated to sample storage time, detectable already after one year. With the use of a linear regression model we could adjust for the storage-dependent effect, leading to the identification of 54 differentially expressed miRNAs in tongue cancer, compared to 16 when using standard normalization, including up-regulation of a novel miRNA, miR-424.

Transcriptional profiling of the peripheral blood response during tularemia.

Tularemia is a febrile disease caused by the highly contagious bacterium Francisella tularensis. We undertook an analysis of the transcriptional response in peripheral blood during the course of ulceroglandular tularemia by use of Affymetrix microarrays comprising 14,500 genes. Samples were obtained from seven individuals at five occasions during 2 weeks after the first hospital visit and convalescent samples 3 months later. In total, 265 genes were differentially expressed, 95 of which at more than one time point. The differential expression was verified with real-time quantitative polymerase chain reaction for 36 genes (R(2)=0.590). The most prominent changes were noted in samples drawn on days 2-3 and a considerable proportion of the upregulated genes appeared to represent an interferon-gamma-induced response and also a proapoptotic response. Genes involved in the generation of innate and acquired immune responses were found to be downregulated, presumably a pathogen-induced event. A logistic regression analysis revealed that seven genes were good predictors of the early phase of tularemia. This is the first description of the transcriptional host response to ulceroglandular tularemia and the study has identified gene subsets relevant to the pathogenesis of the disease and subsets that may serve as early diagnostic biomarkers.

Swelling behavior of the tomato cell wall network.

The swelling of tomato pectin and isolated tomato pericarp cell wall material was investigated in aqueous media under different ionic conditions, pH, and external osmotic stress. Conditions were chosen to include those that would be encountered in vivo. Swelling in these systems was strongly influenced by the polyelectrolyte nature of the polymer and the extent of cross-linking with divalent counterions.

Hydration of pectic polysaccharides.

The hydration and swelling of pectic polysaccharides was examined at different pHs and ionic strengths as a function of osmotic stress. For weakly charged pectic polysaccharides at low concentrations of a monovalent salt (20 mM), the main driving force for swelling originates from a polyelectrolyte effect due to the translational entropy of ions within the film. Swelling is reduced at higher salt concentrations and lower pHs. Polyelectrolyte collapse and minimal swelling is observed for more highly charged pectic polysaccharides. Replacement of the Na(+) counterion with Ca(2+) results in minimal swelling and the formation of network structures even for the weakly charged pectic polysaccharides.

13C MAS NMR studies of the effects of hydration on the cell walls of potatoes and Chinese water chestnuts.

13C NMR with magic angle spinning (MAS) has been employed to investigate the cell walls of potatoes and Chinese water chestnuts over a range of hydration levels. Both single-pulse excitation (SPEMAS) and cross-polarization (CPMAS) experiments were carried out. Hydration led to a substantial increase in signal intensities of galactan and galacturonan in the SPEMAS spectra and a decrease in line width, implying mobilization in the backbone and side chains of pectin. In CPMAS spectra of both samples, noncellulose components showed signal loss as hydration increased. However, the signals of some galacturonan in the 3(1) helix configuration remained in the spectra even when the water content was as high as 110%. Cellulose was unaffected. It is concluded that the pectic polysaccharides experience a distribution of molecular conformations and mobility, whereas cellulose remained as typical rigid solid.

Ileal effluent as a fermentation substrate: implications for butyrate production in the colon.

Fermentation of fiber can lead to an enhanced production of short chain fatty acids (SCFA) and, hence, contribute to the proposed anticarcinogenic properties of butyrate in the colon. The fermentation of fiber isolates and the corresponding ileal effluents has been compared under in vitro conditions. Yield of SCFA per gram of substrate fermented was similar for isolates and fiber-enriched effluents (approximately 4.9 mmol/g) and it could be inferred that nonfiber components of effluent also generated SCFA. Butyrate production was highest for glucan-based polymers (approximately 30% total SCFA) and, from the measured acidogenic profile, production of SCFA will occur mainly in the proximal colon. The buffering capacity of ileal effluents during fermentation restrict the potential for a reduction in pH during acidogenesis compared to fiber isolates. This buffering capacity could limit the bioavailability of butyrate in the colon and, hence, the ability to satisfy the proposed antineoplastic properties of butyrate in the colon.

Characterisation of the binding capacities and affinities of wheat bran, fruit and vegetable fibres for MeIQx, before and after fermentation.

Binding capacities and affinities of dietary fibres and fermented fibre residues for MeIQx are much higher for brans than for fruit and vegetable fibres. Bran residues concentrated distally in the colon would provide a substantial binding matrix. Small intestinal conditions are not so conducive to hydrophobic adsorption.

The effects of pH and bile salts on the binding of MeIQx to wheat bran fibre.

The effects of pH and of the bile salts, sodium cholate, chenodeoxycholate, taurocholate, deoxycholate and glycodeoxycholate on the adsorption of the heterocyclic aromatic amine mutagen and carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) onto wheat bran cell wall material have been examined. The highest binding affinity of MeIQx for bran was at pH 5.5. Binding affinity declined more rapidly with pH > 5.5 than with pH < 5.5. Bile salts in solution did not appreciably affect the binding of MeIQx to bran, but where the bile salts formed a suspension then the adsorption was reduced. Where precipitation of bile salts occurred then the free concentration of MeIQx was also reduced, indicating that MeIQx binding/immobilization could be enhanced through interaction with bile salts.

The consequences of fruit and vegetable fibre fermentation on their binding capacity for MeIQx and the effects of soluble fibre sources on the binding affinity of wheat bran preparations.

Fruits and vegetables provide dietary fibre some of which is partly soluble in the upper gut; in the colon it is highly fermentable. Using alcohol-insoluble residues prepared from a range of fruits and vegetables the effects of fermentation on the changes in composition and binding capacity have been assessed for the hydrophobic mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Fermentation was extensive and resulted in destruction of most of the pectic polysaccharides. Of the unfermented vegetable fibre only cabbage had a measurable hydrophobic binding capacity. The binding capacities of unfermented apple, carrot and sugar beet were negligible. After fermentation, binding capacities, (per mg of fermented residue), increased. Although fermented cabbage was found to have the highest capacity of the fruit and vegetable fibres this remained less than the least effective of unfermented wheat bran samples which had a relatively high affinity for MeIQx. Mucin inhibited the binding of MeIQx to wheat bran fibre but apple fibre did not. The results show that the contribution of fruit and vegetable fibre to a hydrophobic binding matrix in the colon is insignificant and the suggested harmful effect of fruit and vegetable fibre, maintaining hydrophobic mutagens in solution, can be prevented by the presence of wheat bran fibre.

The effect of fibre source and fermentation on the apparent hydrophobic binding properties of wheat bran preparations for the mutagen 2-amino-3,8- dimethylimidazo[4,5-f]quinoxaline (MeIQx).

Preparations of dietary fibre have an ability to bind potentially toxic compounds in vitro, but it is unclear how the measured binding properties are a reflection of either the relative affinity of such compounds for fibre or the saturation binding capacity of the fibre. Cooking and processing of foods and fermentation activity in the colon can result in significant modification of the structure of the fibre matrix. Hence, binding properties measured in vitro may be significantly altered from those at the proposed site of fibre activity. Using cell wall material (CWM) prepared from three distinct wheat fibre sources, the effects of fermentation have been monitored and the consequences on the binding properties assessed. The CWM preparations were feremented for either 0, 6, 18 or 24 h in vitro, using a human faecal inoculum. The coarse bran CWM was also subjected to a simulated gastric treatment. Fermentation resulted in a loss of material from each CWM preparation, the loss being consistent with the bran source, and the maximum extent of fermentation was reached within 18 h. The in vitro binding of the mutagen 2-amino-3,8- dimethylimidazo[4,5-f]quinoxaline (MeIQx) to the unfermented and fermented fibre fractions showed fine bran CWM had a much higher affinity for MeIQx than coarse bran. A 6 h fermentation had little effect on the binding affinity to fine bran, but the affinity for coarse bran became similar to that of the fine bran. Further fermentation for 18 h or 24 h resulted in a further slight increase in the binding affinity for coarse and fine bran, but, more significantly, the binding capacity of each was increased. The binding properties of the beeswing bran were not significantly affected by fermentation. Wheat bran has the potential to bind hydrophobic mutagens in the diet and this potential can be enhanced after fermentation under colonic conditions.

Cell-wall polysaccharides and glycoproteins of parenchymatous tissues of runner bean (Phaseolus coccineus).

1. Polymers were solubilized from the cell walls of parenchyma from mature runner-bean pods with minimum degradation by successive extractions with cyclohexane-trans-1,2-diamine-NNN'N'-tetra-acetate (CDTA), Na2CO3 and KOH to leave the alpha-cellulose residue, which contained cross-linked pectic polysaccharides and Hyp-rich glycoproteins. These were solubilized with chlorite/acetic acid and cellulase. The polymers were fractionated by anion-exchange chromatography, and fractions were subjected to methylation analysis. 2. The pectic polysaccharides differed in their ease of extraction, and a small proportion were highly cross-linked. The bulk of the pectic polysaccharides solubilized by CDTA and Na2CO3 were less branched than those solubilized by KOH. There was good evidence that most of the pectic polysaccharides were not degraded during extraction. 3. The protein-containing fractions included Hyp-rich and Hyp-poor glycoproteins associated with easily extractable pectic polysaccharides, Hyp-rich glycoproteins solubilized with 4M-KOH+borate, the bulk of which were not associated with pectic polysaccharides, and highly cross-linked Hyp-rich glycoproteins. 4. Isodityrosine was not detected, suggesting that it does not have a (major) cross-linking role in these walls. Instead, it is suggested that phenolics, presumably linked to C-5 of 3,5-linked Araf residues of Hyp-rich glycoproteins, serve to cross-link some of the polymers. 5. There were two main types of xyloglucan, with different degrees of branching. The bulk of the less branched xyloglucans were solubilized by more-concentrated alkali. The anomeric configurations of the sugars in one of the highly branched xyloglucans were determined by 13C-n.m.r. spectroscopy. 6. The structural features of the cell-wall polymers and complexes are discussed in relation to the structure of the cell walls of parenchyma tissues.