Gut microbiota maturity mediates the protective effect of siblings on food allergy.

BACKGROUND: The mechanisms underlying the protective effect of older siblings on allergic disease remain unclear but may relate to the infant gut microbiota. OBJECTIVE: We sought to investigate whether having older siblings decreases the risk of IgE-mediated food allergy by accelerating the maturation of the infant gut microbiota. METHODS: In a birth cohort assembled using an unselected antenatal sampling frame (n = 1074), fecal samples were collected at 1 month, 6 months, and 1 year, and food allergy status at 1 year was determined by skin prick test and in-hospital food challenge. We used 16S rRNA gene amplicon sequencing to derive amplicon sequence variants. Among a random subcohort (n = 323), microbiota-by-age z scores at each time point were calculated using fecal amplicon sequence variants to represent the gut microbiota maturation over the first year of life. RESULTS: A greater number of siblings was associated with a higher microbiota-by-age z score at age 1 year (ß  = 0.15 per an additional sibling; 95% CI, 0.05-0.24; P = .003), which was in turn associated with decreased odds of food allergy (odds ratio, 0.45; 95% CI, 0.33-0.61; P < .001). Microbiota-by-age z scores mediated 63% of the protective effect of siblings. Analogous associations were not observed at younger ages. CONCLUSIONS: The protective effect of older siblings on the risk of developing IgE-mediated food allergy during infancy is substantially mediated by advanced maturation of the gut microbiota at age 1 year.

Household size, T regulatory cell development, and early allergic disease: a birth cohort study.

BACKGROUND: Children born to larger households have less allergic disease. T regulatory cell (Treg) development may be a relevant mechanism, but this has not been studied longitudinally. OBJECTIVE: We aim to (i) describe how prenatal and postnatal environmental factors are associated with Treg development and (ii) investigate whether serial Treg measures predict allergic outcomes at 1 year of age. METHODS: A birth cohort (n = 1074) with information on prenatal and postnatal early life factors. Both naïve Treg (nTreg) and activated Treg (aTreg) cell populations (as a proportion of CD4+ T cells) were available in 463 infants at birth (cord blood), 600 at 6 months, and 675 at 12 months. 191 infants had serial measures. Measures of allergic status at 12 months were polysensitization (sensitization to 2 or more allergens), clinically proven food allergy, atopic eczema, and atopic wheeze. RESULTS: Infants born to larger households (3 or more residents) had higher longitudinal nTreg proportions over the first postnatal year with a mean difference (MD) of 0.67 (95% CI 0.30-1.04)%. Higher nTreg proportions at birth were associated with a reduced risk of infant allergic outcomes. Childcare attendance and breastfeeding were associated with higher longitudinal nTreg proportions (MD 0.48 (95% CI 0.08-0.80)%. CONCLUSION: Multiple prenatal and postnatal microbial factors are associated with nTreg and aTreg development. Larger household size was associated with higher nTreg at birth which in turn was associated with reduced allergic sensitization and disease at 12 months of age.

The maternal gut microbiome during pregnancy and offspring allergy and asthma.

Environmental exposures during pregnancy that alter both the maternal gut microbiome and the infant's risk of allergic disease and asthma include a traditional farm environment and consumption of unpasteurized cow's milk, antibiotic use, dietary fiber, and psychosocial stress. Multiple mechanisms acting in concert may underpin these associations and prime the infant to acquire immune competence and homeostasis following exposure to the extrauterine environment. Cellular and metabolic products of the maternal gut microbiome can promote the expression of microbial pattern recognition receptors, as well as thymic and bone marrow hematopoiesis relevant to regulatory immunity. At birth, transmission of maternally derived bacteria likely leverages this in utero programming to accelerate postnatal transition from a TH2- to TH1- and TH17-dominant immune phenotype and maturation of regulatory immune mechanisms, which in turn reduce the child's risk of allergic disease and asthma. Although our understanding of these phenomena is rapidly evolving, the field is relatively nascent, and we are yet to translate existing knowledge into interventions that substantially reduce disease risk in humans. Here, we review evidence that the maternal gut microbiome impacts the offspring's risk of allergic disease and asthma, discuss challenges and future directions for the field, and propose the hypothesis that maternal carriage of Prevotella copri during pregnancy decreases the offspring's risk of allergic disease via production of succinate, which in turn promotes bone marrow myelopoiesis of dendritic cell precursors in the fetus.

Shortened Infant Telomere Length Is Associated with Attention Deficit/Hyperactivity Disorder Symptoms in Children at Age Two Years: A Birth Cohort Study.

Environmental factors can accelerate telomere length (TL) attrition. Shortened TL is linked to attention deficit/hyperactivity disorder (ADHD) symptoms in school-aged children. The onset of ADHD occurs as early as preschool-age, but the TL-ADHD association in younger children is unknown. We investigated associations between infant TL and ADHD symptoms in children and assessed environmental factors as potential confounders and/or mediators of this association. Relative TL was measured by quantitative polymerase chain reaction in cord and 12-month blood in the birth cohort study, the Barwon Infant Study. Early life environmental factors collected antenatally to two years were used to measure confounding. ADHD symptoms at age two years were evaluated by the Child Behavior Checklist Attention Problems (AP) and the Attention Deficit/Hyperactivity Problems (ADHP). Associations between early life environmental factors on TL or ADHD symptoms were assessed using multivariable regression models adjusted for relevant factors. Telomere length at 12 months (TL12), but not at birth, was inversely associated with AP (ß = -0.56; 95% CI (-1.13, 0.006); p = 0.05) and ADHP (ß = -0.66; 95% CI (-1.11, -0.21); p = 0.004). Infant secondhand smoke exposure at one month was independently associated with shorter TL12 and also higher ADHD symptoms. Further work is needed to elucidate the mechanisms that influence TL attrition and early neurodevelopment.

Burden of infection in Australian infants.

AIM: To determine the incidence, risk factors and health service utilisation for infection in the first 12 months of life in a population-derived Australian pre-birth cohort. METHODS: The Barwon Infant Study is a population-derived pre-birth cohort with antenatal recruitment (n = 1074) based in Geelong, Victoria, Australia. Infection data were collected by parent report, and general practitioner and hospital records at 1, 3, 6, 9 and 12 months of age. We calculated the incidence of infection, attendance at a health service with infection and used multiple negative binomial regression to investigate the effects of a range of exposures on incidence of infection. RESULTS: In the first 12 months of life, infections of the upper and lower respiratory tract (henceforth 'respiratory infections'), conjunctivitis and gastroenteritis occurred at a rate of 0.35, 0.04 and 0.04 episodes per child-month, respectively. A total of 482 (72.4%) infants attended a general practitioner with an infection and 69 (10.4%) infants attended the emergency department. Maternal antibiotic exposure in pregnancy and having older siblings were associated with respiratory infection. Childcare attendance by 12 months of age was associated with respiratory infections and gastroenteritis. Breastfeeding, even if less than 4 weeks in total, was associated with reduced respiratory infection. CONCLUSION: Infection, especially of the respiratory tract, is a common cause of morbidity in Australian infants. Several potentially modifiable risk factors were identified, particularly for respiratory infections. Most infections were managed by general practitioners and 1 in 10 infants attended an emergency department with infection in the first year of life.

Deserters on the atopic march: Risk factors, immune profile and clinical outcomes of food sensitized-tolerant infants.

BACKGROUND: A few studies have investigated the antecedents and outcomes of infants who demonstrate IgE sensitization to foods that they clinically tolerate. Improved understanding of this sensitized-tolerant phenotype may inform strategies for the prevention of food allergy. METHODS: In an Australian birth cohort (n = 1074), assembled using an unselected antenatal sampling frame, participants were categorized as nonsensitized (NS), sensitizedtolerant (ST), or food allergic (FA) based on skin prick testing and food challenge at 12 months of age. Environmental exposures were recorded throughout. Cord blood regulatory T-cell populations were measured at birth. Subsequent childhood allergic disease was assessed by parent report, clinical examination, and repeat skin prick testing. RESULTS: The covariates of interest varied between NS (n = 698), ST (n = 27), and FA (n = 61) groups as follows, suggesting that across these measures, the ST group was more similar to the NS than the FA group: family history of eczema NS 44.6%, ST. 44.6%, FA 65.6%; pet ownership at 12 months: NS 71.5%, ST 81.5%, FA 45.8%; eczema during the first 12 months: NS 19%, ST 32%, FA 64%; and aeroallergen sensitization at 4 years: NS 19.1%, ST 28.6%, FA 44.4%. At birth, a higher proportion of activated regulatory T cells was associated with ST (OR = 2.89, 95% CI 1.03-8.16, P = .045). CONCLUSION: Food-sensitized-tolerance in infancy appears to be associated with a similar pattern of exposures, immunity, and outcomes to nonsensitized infants. In addition, we found some evidence that an elevated proportion of activated regulatory T cells at birth was specific to the sensitized-tolerant infants, which may be relevant to suppression of clinical disease.

Naïve regulatory T cells in infancy: Associations with perinatal factors and development of food allergy.

BACKGROUND: In previous studies, deficits in regulatory T-cell (Treg) number and function at birth have been linked with subsequent allergic disease. However, longitudinal studies that account for relevant perinatal factors are required. The aim of this study was to investigate the relationship between perinatal factors, naïve Treg (nTreg) over the first postnatal year and development of food allergy. METHODS: In a birth cohort (n = 1074), the proportion of nTreg in the CD4+ T-cell compartment was measured by flow cytometry at birth (n = 463), 6 (n = 600) and 12 (n = 675) months. IgE-mediated food allergy was determined by food challenge at 1 year. Associations between perinatal factors (gestation, labour, sex, birth size), nTreg at each time point and food allergy at 1 year were examined by linear regression. RESULTS: A higher proportion of nTreg at birth, larger birth size and male sex was each associated with higher nTreg in infancy. Exposure to labour, as compared to delivery by prelabour Caesarean section, was associated with a transient decrease nTreg. Infants that developed food allergy had decreased nTreg at birth, and the labour-associated decrease in nTreg at birth was more evident among infants with subsequent food allergy. Mode of birth was not associated with risk of food allergy, and there was no evidence that nTreg at either 6 or 12 months were related to food allergy. CONCLUSION: The proportion of nTreg at birth is a major determinant of the proportion present throughout infancy, highlighting the importance of prenatal immune development. Exposure to the inflammatory stimulus of labour appears to reveal differences in immune function among infants at risk of food allergy.

High incidence of respiratory disease in Australian infants despite low rate of maternal cigarette smoking.

AIM: The burden of wheezing illnesses in Australian infants has not been documented since the success of initiatives to reduce maternal cigarette smoking. We aimed to determine the incidence of wheeze and related health-care utilisation during the first year of life among a contemporary Australian birth cohort. METHODS: A birth cohort of 1074 infants was assembled between 2010 and 2013. Parents completed questionnaires periodically. Several non-exclusive infant respiratory disease phenotypes were defined, including any wheeze, wheeze with shortness of breath and recurrent wheeze. Skin prick testing was performed to determine atopic wheeze. Health-care utilisation for respiratory disease was determined from questionnaires and hospital medical records. RESULTS: Retention to 1 year was 840/1074 (83%). The incidence of any wheeze was 51.8% (95% confidence interval (CI) 48.3-55.2%), wheeze with shortness of breath 20.6% (95% CI 17.9-23.5), recurrent wheeze 19.4% (95% CI 16.8-22.2) and atopic wheeze 6% (95% CI 4.6-7.8). Respiratory illness resulted in primary health-care utilisation in 82.2% (95% CI 79.3-84.8) of participants and hospital presentation in 8.8% (95% CI 7.2-10.6). Maternal smoking during pregnancy was uncommon (15.7%) and was not associated with wheeze or health resource utilisation. Male gender, familial atopy and asthma and smaller household size were associated with a higher incidence of wheeze. CONCLUSIONS: The incidence of wheezing illness among Australian infants remains high despite relatively low rates of maternal smoking during pregnancy. The majority of the health-care burden is borne by primary health-care services. Further research is required to inform novel prevention strategies.

A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity.

The discovery that theMPC1andMPC2genes encode the protein components of the mitochondrial pyruvate carrier (MPC) has invigorated studies of mitochondrial pyruvate transport and its regulation in normal and disease states. Indeed, recent reports have demonstrated MPC involvement in the control of cell fate in cancer and gluconeogenesis in models of type 2 diabetes. Biochemical measurements of MPC activity are foundational for understanding the role of pyruvate transport in health and disease. We developed a 96-well scaled method of [(14)C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPCKm(28.0 ± 3.9 µm) andVmax(1.08 ± 0.05 nmol/min/mg), which have not previously been reported.KmandVmaxof the rat liver MPC were found to be 71.2 ± 17 µmand 1.42 ± 0.14 nmol/min/mg, respectively. Additionally, we performed parallel pyruvate uptake and oxidation experiments with the same biological samples and show differential results in response to fasting, demonstrating the continued importance of a direct MPC activity assay. We expect this method will be of value for understanding the contribution of the MPC activity to health and disease states where pyruvate metabolism is expected to play a prominent role.

Primary amoebic meningoencephalitis in North Queensland: the paediatric experience.

Primary amoebic meningoencephalitis (PAM) is a fulminant, diffuse haemorrhagic meningoencephalitis caused by Naegleria fowleri, with an almost invariably fatal outcome. In Australia and the developed world, PAM remains a rare disease, although it is very likely that large numbers of cases go undetected in developing countries. N. fowleri is a thermophilic, free-living amoeba with a worldwide distribution. It is acquired when contaminated fresh water is flushed into the nose and penetrates the central nervous system via the cribriform plate. Clinical features are similar to those of bacterial meningitis, but it does not respond to standard therapy and rapid progression to death occurs in most cases. Some survivors have been reported; these patients received early treatment with amphotericin B in combination with a variety of other medications. Our review describes the local and worldwide experience of this disease and its clinical features, and discusses the associated diagnostic challenges. We hope that by detailing the local response to a recent case, and the outcomes of our public health campaign, we can improve the knowledge of this rare disease for doctors working in rural and remote Australia.

Regulation of pyruvate metabolism and human disease.

Pyruvate is a keystone molecule critical for numerous aspects of eukaryotic and human metabolism. Pyruvate is the end-product of glycolysis, is derived from additional sources in the cellular cytoplasm, and is ultimately destined for transport into mitochondria as a master fuel input undergirding citric acid cycle carbon flux. In mitochondria, pyruvate drives ATP production by oxidative phosphorylation and multiple biosynthetic pathways intersecting the citric acid cycle. Mitochondrial pyruvate metabolism is regulated by many enzymes, including the recently discovered mitochondria pyruvate carrier, pyruvate dehydrogenase, and pyruvate carboxylase, to modulate overall pyruvate carbon flux. Mutations in any of the genes encoding for proteins regulating pyruvate metabolism may lead to disease. Numerous cases have been described. Aberrant pyruvate metabolism plays an especially prominent role in cancer, heart failure, and neurodegeneration. Because most major diseases involve aberrant metabolism, understanding and exploiting pyruvate carbon flux may yield novel treatments that enhance human health.

Near-infrared fluorescent sensor for in vivo copper imaging in a murine Wilson disease model.

Copper is an essential metal nutrient that is tightly regulated in the body because loss of its homeostasis is connected to severe diseases such as Menkes and Wilson diseases, Alzheimer's disease, prion disorders, and amyotrophic lateral sclerosis. The complex relationships between copper status and various stages of health and disease remain challenging to elucidate, in part due to a lack of methods for monitoring dynamic changes in copper pools in whole living organisms. Here we present the synthesis, spectroscopy, and in vivo imaging applications of Coppersensor 790, a first-generation fluorescent sensor for visualizing labile copper pools in living animals. Coppersensor 790 combines a near-infrared emitting cyanine dye with a sulfur-rich receptor to provide a selective and sensitive turn-on response to copper. This probe is capable of monitoring fluctuations in exchangeable copper stores in living cells and mice under basal conditions, as well as in situations of copper overload or deficiency. Moreover, we demonstrate the utility of this unique chemical tool to detect aberrant increases in labile copper levels in a murine model of Wilson disease, a genetic disorder that is characterized by accumulation of excess copper. The ability to monitor real-time copper fluxes in living animals offers potentially rich opportunities to examine copper physiology in health and disease.

Transport kinetics and selectivity of HpUreI, the urea channel from Helicobacter pylori.

Helicobacter pylori's unique ability to colonize and survive in the acidic environment of the stomach is critically dependent on uptake of urea through the urea channel, HpUreI. Hence, HpUreI may represent a promising target for the development of specific drugs against this human pathogen. To obtain insight into the structure-function relationship of this channel, we developed conditions for the high-yield expression and purification of stable recombinant HpUreI. Detergent-solubilized HpUreI forms a homotrimer, as determined by chemical cross-linking. Urea dissociation kinetics of purified HpUreI were determined by means of the scintillation proximity assay, whereas urea efflux was measured in HpUreI-containing proteoliposomes using stopped-flow spectrometry to determine the kinetics and selectivity of the urea channel. The kinetic analyses revealed that urea conduction in HpUreI is pH-sensitive and saturable with a half-saturation concentration (or K(0.5)) of ~163 mM. The extent of binding of urea by HpUreI was increased at lower pH; however, the apparent affinity of urea binding (~150 mM) was not significantly pH-dependent. The solute selectivity analysis indicated that HpUreI is highly selective for urea and hydroxyurea. Removing either amino group of urea molecules diminishes their permeability through HpUreI. Similar to urea conduction, diffusion of water through HpUreI is pH-dependent with low water permeability at neutral pH.

Wilson disease at a single cell level: intracellular copper trafficking activates compartment-specific responses in hepatocytes.

Wilson disease (WD) is a severe hepato-neurologic disorder that affects primarily children and young adults. WD is caused by mutations in ATP7B and subsequent copper overload. However, copper levels alone do not predict severity of the disease. We demonstrate that temporal and spatial distribution of copper in hepatocytes may play an important role in WD pathology. High resolution synchrotron-based x-ray fluorescence imaging in situ indicates that copper does not continuously accumulate in Atp7b(-/-) hepatocytes, but reaches a limit at 90-300 fmol. The lack of further accumulation is associated with the loss of copper transporter Ctr1 from the plasma membrane and the appearance of copper-loaded lymphocytes and extracellular copper deposits. The WD progression is characterized by changes in subcellular copper localization and transcriptome remodeling. The synchrotron-based x-ray fluorescence imaging and mRNA profiling both point to the key role of nucleus in the initial response to copper overload and suggest time-dependent sequestration of copper in deposits as a protective mechanism. The metabolic pathways, up-regulated in response to copper, show compartmentalization that parallels changes in subcellular copper concentration. In contrast, significant down-regulation of lipid metabolism is observed at all stages of WD irrespective of copper distribution. These observations suggest new stage-specific as well as general biomarkers for WD. The model for the dynamic role of copper in WD is proposed.

Systems biology approach to Wilson's disease.

Wilson's disease (WD) is a severe disorder of copper misbalance, which manifests with a wide spectrum of liver pathology and/or neurologic and psychiatric symptoms. WD is caused by mutations in a gene encoding a copper-transporting ATPase ATP7B and is accompanied by accumulation of copper in tissues, especially in the liver. Copper-chelation therapy is available for treatment of WD symptoms and is often successful, however, significant challenges remain with respect to timely diagnostics and treatment of the disease. The lack of genotype-phenotype correlation remains unexplained, the causes of fulminant liver failure are not known, and the treatment of neurologic symptoms is only partially successful, underscoring the need for better understanding of WD mechanisms and factors that influence disease manifestations. Recent gene and protein profiling studies in animal models of WD began to uncover cellular processes that are primarily affected by copper accumulation in the liver. The results of such studies, summarized in this review, revealed new molecular players and pathways (cell cycle and cholesterol metabolism, mRNA splicing and nuclear receptor signaling) linked to copper misbalance. A systems biology approach promises to generate a comprehensive view of WD onset and progression, thus helping with a more fine-tune treatment and monitoring of the disorder.

Innate Immune Activation and Circulating Inflammatory Markers in Preschool Children.

Early childhood is characterised by repeated infectious exposures that result in inflammatory responses by the innate immune system. In addition, this inflammatory response to infection is thought to contribute to the epidemiological evidence linking childhood infection and adult non-communicable diseases. Consequently, the relationship between innate immune responses and inflammation during early life may inform prevention of NCDs later in life. In adults, non-genetic host factors such as age, sex, and obesity, strongly impact cytokine production and circulating mediators, but data in children are lacking. Here, we assessed cytokine responses and inflammatory markers in a population of healthy preschool children (mean age 4.2 years). We studied associations between cytokines, plasma inflammatory markers and non-genetic host factors, such as sex, age, adiposity, season, and immune cell composition. Similar to adults, boys had a higher inflammatory response than girls, with IL-12p70 and IL-10 upregulated following TLR stimulation. Adiposity and winter season were associated with increased circulating inflammatory markers but not cytokine production. The inflammatory markers GlycA and hsCRP were positively associated with production of a number of cytokines and may therefore reflect innate immune function and inflammatory potential. This dataset will be informative for future prospective studies relating immune parameters to preclinical childhood NCD phenotypes.