Maternal gut bacteria drive intestinal inflammation in offspring with neurodevelopmental disorders by altering the chromatin landscape of CD4+ T cells.

Children with autism spectrum disorders often display dysregulated immune responses and related gastrointestinal symptoms. However, the underlying mechanisms leading to the development of both phenotypes have not been elucidated. Here, we show that mouse offspring exhibiting autism-like phenotypes due to prenatal exposure to maternal inflammation were more susceptible to developing intestinal inflammation following challenges later in life. In contrast to its prenatal role in neurodevelopmental phenotypes, interleukin-17A (IL-17A) generated immune-primed phenotypes in offspring through changes in the maternal gut microbiota that led to postnatal alterations in the chromatin landscape of naive CD4+ T cells. The transfer of stool samples from pregnant mice with enhanced IL-17A responses into germ-free dams produced immune-primed phenotypes in offspring. Our study provides mechanistic insights into why children exposed to heightened inflammation in the womb might have an increased risk of developing inflammatory diseases in addition to neurodevelopmental disorders.

Role of Maternal Microbiota and Nutrition in Early-Life Neurodevelopmental Disorders.

The rise in the prevalence of obesity and other related metabolic diseases has been paralleled by an increase in the frequency of neurodevelopmental problems, which has raised the likelihood of a link between these two phenomena. In this scenario, maternal microbiota is a possible linking mechanistic pathway. According to the "Developmental Origins of Health and Disease" paradigm, environmental exposures (in utero and early life) can permanently alter the body's structure, physiology, and metabolism, increasing illness risk and/or speeding up disease progression in offspring, adults, and even generations. Nutritional exposure during early developmental stages may induce susceptibility to the later development of human diseases via interactions in the microbiome, including alterations in brain function and behavior of offspring, as explained by the gut-brain axis theory. This review provides an overview of the implications of maternal nutrition on neurodevelopmental disorders and the establishment and maturation of gut microbiota in the offspring.

Dissecting the contribution of host genetics and the microbiome in complex behaviors.

The core symptoms of many neurological disorders have traditionally been thought to be caused by genetic variants affecting brain development and function. However, the gut microbiome, another important source of variation, can also influence specific behaviors. Thus, it is critical to unravel the contributions of host genetic variation, the microbiome, and their interactions to complex behaviors. Unexpectedly, we discovered that different maladaptive behaviors are interdependently regulated by the microbiome and host genes in the Cntnap2-/- model for neurodevelopmental disorders. The hyperactivity phenotype of Cntnap2-/- mice is caused by host genetics, whereas the social-behavior phenotype is mediated by the gut microbiome. Interestingly, specific microbial intervention selectively rescued the social deficits in Cntnap2-/- mice through upregulation of metabolites in the tetrahydrobiopterin synthesis pathway. Our findings that behavioral abnormalities could have distinct origins (host genetic versus microbial) may change the way we think about neurological disorders and how to treat them.

Is there a dysbiosis in individuals with a neurodevelopmental disorder compared to controls over the course of development? A systematic review.

Many scientific papers reported that an unbalanced gut microbiota could lead to or worsen neurodevelopmental disorders (NDD). A dysbiosis may then be observed in the course of development and mark a dysfunction within what is called the gut-brain axis. The aim of this systematic review is to investigate potential evidence of dysbiosis in children and young adults with NDD compared to controls. Using the PRISMA guidelines we systematically reviewed studies that compared the gut microbiota in NDD participants (with an age inferior to thirty) to the gut microbiota of controls, regardless of the data analysis methods used. The MEDLINE, Scopus and PsycINFO databases were searched up to September 2018. 31 studies with a total sample size of 3002 ASD (Autism Spectrum Disorder) and 84 ADHD (Attention Deficit Hyperactivity Disorder) participants were included in this systematic review. Independent data extraction and quality assessment were conducted. The quality of the studies was rated from low to high. Population characterization and experimental methods were highly heterogeneous in terms of the data available, selection of criteria, and dysbiosis measurement. A dysbiosis was reported in 28 studies in terms of either diversity, bacterial composition or metabolome dysfunction. Due to heterogeneity, a quantitative synthesis was not applicable. In this paper, we discuss the different biases to understand the complexity of microbiota and neurodevelopmental disorders to provide leads for future cohort studies looking to answer the questions raised by the trillions of microorganisms that inhabit key body niches.

Assessment of Neonatal Intensive Care Unit Practices and Preterm Newborn Gut Microbiota and 2-Year Neurodevelopmental Outcomes.

Importance: In very preterm newborns, gut microbiota is highly variable with major dysbiosis. Its association with short-term health is widely studied, but the association with long-term outcomes remains unknown. Objective: To investigate in preterm newborns the associations among practice strategies in neonatal intensive care units (NICUs), gut microbiota, and outcomes at 2 years. Design, Setting, and Participants: EPIFLORE is a prospective observational cohort study that includes a stool sample collection during the fourth week after birth. Preterm newborns of less than 32 weeks of gestational age (GA) born in 2011 were included from 24 NICUs as part of the French nationwide population-based cohort, EPIPAGE 2. Data were collected from May 2011 to December 2011 and analyzed from September 2016 to December 2018. Exposures: Eight NICU strategies concerning sedation, ventilation, skin-to-skin practice, antibiotherapy, ductus arteriosus, and breastfeeding were assessed. A NICU was considered favorable to a practice if the percentage of that practice in the NICU was more than the expected percentage. Main Outcomes and Measures: Gut microbiota was analyzed by 16S ribosomal RNA gene sequencing and characterized by a clustering-based method. The 2-year outcome was defined by death or neurodevelopmental delay using a Global Ages and Stages questionnaire score. Results: Of 577 newborns included in the study, the mean (SD) GA was 28.3 (2.0) weeks, and 303 (52.5%) were male. Collected gut microbiota was grouped into 5 discrete clusters. A sixth cluster included nonamplifiable samples owing to low bacterial load. Cluster 4 (driven by Enterococcus [n = 63]), cluster 5 (driven by Staphylococcus [n = 52]), and cluster 6 (n = 93) were significantly associated with lower mean (SD) GA (26.7 [1.8] weeks and 26.8 [1.9] weeks, respectively) and cluster 3 (driven by Escherichia/Shigella [n = 61]) with higher mean (SD) GA (29.4 [1.6] weeks; P = .001). Cluster 3 was considered the reference. After adjustment for confounders, no assisted ventilation at day 1 was associated with a decreased risk of belonging to cluster 5 or cluster 6 (adjusted odds ratio [AOR], 0.21 [95% CI, 0.06-0.78] and 0.19 [95% CI, 0.06-0.62], respectively) when sedation (AOR, 10.55 [95% CI, 2.28-48.87] and 4.62 [1.32-16.18], respectively) and low volume of enteral nutrition (AOR, 10.48 [95% CI, 2.48-44.29] and 7.28 [95% CI, 2.03-26.18], respectively) was associated with an increased risk. Skin-to-skin practice was associated with a decreased risk of being in cluster 5 (AOR, 0.14 [95% CI, 0.04-0.48]). Moreover, clusters 4, 5, 6 were significantly associated with 2-year nonoptimal outcome (AOR, 6.17 [95% CI, 1.46-26.0]; AOR, 4.53 [95% CI, 1.02-20.1]; and AOR, 5.42 [95% CI, 1.36-21.6], respectively). Conclusions and Relevance: Gut microbiota of very preterm newborns at week 4 is associated with NICU practices and 2-year outcomes. Microbiota could be a noninvasive biomarker of immaturity.

Gut Microbiota Dysbiosis Associated With Altered Production of Short Chain Fatty Acids in Children With Neurodevelopmental Disorders.

While gut microbiota dysbiosis has been linked with autism, its role in the etiology of other neurodevelopmental disorders (NDD) is largely underexplored. To our knowledge this is the first study to evaluate gut microbiota diversity and composition in 36 children from the Republic of Serbia diagnosed with NDD and 28 healthy children. The results revealed an increased incidence of potentially harmful bacteria, closely related to Clostridium species, in the NDD patient group compared to the Control group: Desulfotomaculum guttoideum (P < 0.01), Intestinibacter bartlettii (P < 0.05), and Romboutsia ilealis (P < 0.001). On the other hand, significantly lower diversity of common commensal bacteria in the NDD group of patients was noticed. Enterococcus faecalis (P < 0.05), Enterococcus gallinarum (P < 0.01), Streptococcus pasteurianus (P < 0.05), Lactobacillus rhamnosus (P < 0.01) and Bifidobacteria sp. were detected in lower numbers of patients or were even absent in some NDD patients. In addition, butyrate-producing bacteria Faecalibacterium prausnitzii (P < 0.01), Butyricicoccus pullicaecorum (P < 0.05), and Eubacterium rectale (P = 0.07) were less frequent in the NDD patient group. In line with that, the levels of fecal short chain fatty acids (SCFAs) were determined. Although significant differences in SCFA levels were not detected between NDD patients and the Control group, a positive correlation was noted between number of rDNA amplicons obtained with universal primers and level of propionic acid, as well as a trend for levels of total SCFAs and butyric acid in the Control group. This correlation is lost in the NDD patient group, indicating that NDD patients' microbiota differs from the microbiota of healthy children in the presence or number of strong SCFA-producing bacteria. According to a range-weighted richness index it was observed that microbial diversity was significantly lower in the NDD patient group. Our study reveals that the intestinal microbiota from NDD patients differs from the microbiota of healthy children. It is hypothesized that early life microbiome might have an impact on GI disturbances and accompanied behavioral problems frequently observed in patients with a broad spectrum of NDD.

Gut microbiota from persons with attention-deficit/hyperactivity disorder affects the brain in mice.

BACKGROUND: The impact of the gut microbiota on host physiology and behavior has been relatively well established. Whether changes in microbial composition affect brain structure and function is largely elusive, however. This is important as altered brain structure and function have been implicated in various neurodevelopmental disorders, like attention-deficit/hyperactivity disorder (ADHD). We hypothesized that gut microbiota of persons with and without ADHD, when transplanted into mice, would differentially modify brain function and/or structure. We investigated this by colonizing young, male, germ-free C57BL/6JOlaHsd mice with microbiota from individuals with and without ADHD. We generated and analyzed microbiome data, assessed brain structure and function by magnetic resonance imaging (MRI), and studied mouse behavior in a behavioral test battery. RESULTS: Principal coordinate analysis showed a clear separation of fecal microbiota of mice colonized with ADHD and control microbiota. With diffusion tensor imaging, we observed a decreased structural integrity of both white and gray matter regions (i.e., internal capsule, hippocampus) in mice that were colonized with ADHD microbiota. We also found significant correlations between white matter integrity and the differentially expressed microbiota. Mice colonized with ADHD microbiota additionally showed decreased resting-state functional MRI-based connectivity between right motor and right visual cortices. These regions, as well as the hippocampus and internal capsule, have previously been reported to be altered in several neurodevelopmental disorders. Furthermore, we also show that mice colonized with ADHD microbiota were more anxious in the open-field test. CONCLUSIONS: Taken together, we demonstrate that altered microbial composition could be a driver of altered brain structure and function and concomitant changes in the animals' behavior. These findings may help to understand the mechanisms through which the gut microbiota contributes to the pathobiology of neurodevelopmental disorders. Video abstract.

Annual Research Review: Critical windows - the microbiota-gut-brain axis in neurocognitive development.

The gut microbiota is a vast, complex, and fascinating ecosystem of microorganisms that resides in the human gastrointestinal tract. As an integral part of the microbiota-gut-brain axis, it is now being recognized that the microbiota is a modulator of brain and behavior, across species. Intriguingly, periods of change in the microbiota coincide with the development of other body systems and particularly the brain. We hypothesize that these times of parallel development are biologically relevant, corresponding to 'sensitive periods' or 'critical windows' in the development of the microbiota-gut-brain axis. Specifically, signals from the microbiota during these periods are hypothesized to be crucial for establishing appropriate communication along the axis throughout the life span. In other words, the microbiota is hypothesized to act like an expected input to calibrate the development of the microbiota-gut-brain axis. The absence or disruption of the microbiota during specific developmental windows would therefore be expected to have a disproportionate effect on specific functions or potentially for regulation of the system as a whole. Evidence for microbial modulation of neurocognitive development and neurodevelopmental risk is discussed in light of this hypothesis, finishing with a focus on the challenges that lay ahead for the future study of the microbiota-gut-brain axis during development.

Lactobacillus rescues postnatal neurobehavioral and microglial dysfunction in a model of maternal microbiome dysbiosis.

Increasing reports of pregnancy events leading to maternal microbiome dysbiosis (MMD) show strong correlates with atypical neurodevelopmental outcomes. However, the mechanism(s) driving microbiome-mediated behavioral dysfunction in offspring remain understudied. Here, we demonstrate the presence of a novel gut commensal bacterium strain, Lactobacillus murinus HU-1, was sufficient to rescue behavioral deficits and brain region-specific microglial activationobserved in MMD-reared murine offspring. We furtheridentified a postnatal window of susceptibility that could prevent social impairments with timed maternal administration of the symbiotic bacterium. Moreover, MMD increased expression of microglial senescence genes, Trp53 and Il1ß, and Cx3cr1 protein in the prefrontal cortex, which correlated with dysfunctional modeling of synapses and accompanied dysbiosis-induced microglial activation. MMD male offspring harboring Lactobacillus murinus HU-1 or lacking Cx3cr1 showed amelioration of these effects. The current study describes a new avenue of influence by which maternally transferred Lactobacillus drives proper development of social behavior in the offspring through microglia-specific regulation of Cx3cr1 signaling.

Addressing safety concerns of probiotic use in preterm babies.

More than 10,000 preterm babies worldwide have been enrolled in trials evaluating probiotics administration for the prevention of necrotising enterocolitis, with very few adverse events reported. Despite this, probiotic safety is frequently cited as a concern when using this intervention. This review addresses why a preterm baby may be at risk when administered a live microbial product, short- and longer-term safety data in relation to probiotic use and regulatory aspects around probiotic manufacture and preparations.

Microbiome programming of brain development: implications for neurodevelopmental disorders.

During the last decade, research on germ-free mice has discovered that the gut microbiome (i.e. the normal bacteria colonizing the gastrointestinal tract) can programme brain function and behaviour during early development. At the same time a growing number of clinical studies have shown altered gut microflora in children with autism spectrum disorder (ASD), in combination with altered bacterial metabolites and inflammatory cytokines being part of the gut-brain axis. This review covers the concept of the microbiome; how it is established during childhood; how it is affected by malnutrition; how it can programme the development of the brain through epigenetic mechanisms; which pathways are used from the gut to the brain; and assesses findings that suggest the gut microbiome may be involved in ASD and other neurodevelopmental disorders. This is a new research field with a number of exciting, but so far fragmented, findings indicating the important role of the normal microbiome in shaping the brain. Research also suggests that disruptions of the microbiome may be involved in the aetiology of neurodevelopmental disorders. WHAT THIS PAPER ADDS: The gut microbiome shapes the brain via the gut-brain axis. The microbiome may play a role in neurodevelopmental disorders.

PROGRAMACIÓN MICROBIAMA DEL DESARROLLO CEREBRAL: IMPLICACIONES PARA LOS TRASTORNOS DEL DESARROLLO NEUROLÓGICO: Durante la última década, la investigación en ratones libres de gérmenes ha descubierto que el microbioma intestinal (es decir, las bacterias normales que colonizan el tracto gastrointestinal) pueden programar la función y el comportamiento cerebral durante el desarrollo temprano. Al mismo tiempo, un número creciente de estudios clínicos ha demostrado una microflora intestinal alterada en niños con trastorno del espectro autista (TEA), en combinación con metabolitos bacterianos alterados y citoquinas inflamatorias que forman parte del eje cerebro-intestino. Esta revisión cubre el concepto de microbioma, incluye; cómo se establece durante la infancia; cómo se ve afectado por la desnutrición; cómo puede programar el desarrollo del cerebro a través de mecanismos epigenéticos; qué vías se utilizan desde el intestino hasta el cerebro; y evalúa los hallazgos que sugieren que el microbioma intestinal puede estar involucrado en el TEA y otros trastornos del desarrollo neurológico. Este es un nuevo campo de investigación con una serie de resultados interesantes, pero hasta ahora fragmentados, que indican el importante papel que desempeña el microbioma normal en la configuración del cerebro. La investigación también sugiere que las alteraciones del microbioma pueden estar involucradas en la etiología de los trastornos del desarrollo neurológico.

PROGRAMAÇÃO DO MICROBIOMA PARA O DESENVOLVIMENTO CEREBRAL: IMPLICAÇÕES PARA TRANSTORNOS DESENVOLVIMENTAIS: Durante a última década, pesquisas em camundongos livres de germes descobriram que o microbioma do sistema gastrointestinal (ou seja, as bactérias que normalmente colonizam o trato gastrointestinal) podem programar a função cerebral e o comportamento no desenvolvimento preococe. Ao mesom tempo, um número crescent de estudos clínicos tem mostrado uma microflora gastrointestinal alterada em crianças com transtorno do espectro autista (TEA), em combinação com metabólitos bacterianos alterados e citocinas inflamatórias sendo parte do eixo gastrointestinal-cérebro. Esta revisão cobre o conceito de microbioma; como ele se estabelece na infância; como é afetado pela malnutrição; como pode programar o desenvolvimento do cérebro por meio de mecanismos epigenéticos; quais vias são usadas do sistema gastrointestinal para o cérebro; e avalia achados que sugerem que o microbioma gastrointestinal pode estar envolvido no TEA e outras desordens neurodesenvolvimentais. Este é um novo campos de pesquisas, porém até o momento, com achados fragmentados indicando o importante papel do miocrobioma normal na formação do cérebro. Pesquisas também sugerem que rupturas no microbioma podem estar envolvidas na etiologia de transtornos neurodesenvolvimentais.

The human-microbiome superorganism and its modulation to restore health.

Microbiome is the community of microorganism that co-live with a host. The human being is the result of the integration of its genome and the coexistence with millions of microorganisms throughout its evolutionary history. Human-microbiome association can be considered a step of integration in evolution, constituting a superorganism. Many emergent diseases are related to the loss of part of this microbiome and its restoration can be achieved by different strategies. Gut microbiome imbalance is particularly associated with numerous inflammatory, immune and nervous system-related diseases by a communication pathway called microbiome-brain axis. Modulation of microbiome by administering prebiotics, like arabinoxylans, and synbiotics is a plausible treatment for dysbiosis, the regulation of neurotransmitters and alleviation of neurological manifestations.

Microbiome-microglia connections via the gut-brain axis.

Microglia, the resident immune cells in the brain, are essential for modulating neurogenesis, influencing synaptic remodeling, and regulating neuroinflammation by surveying the brain microenvironment. Microglial dysfunction has been implicated in the onset and progression of several neurodevelopmental and neurodegenerative diseases; however, the multitude of factors and signals influencing microglial activity have not been fully elucidated. Microglia not only respond to local signals within the brain but also receive input from the periphery, including the gastrointestinal (GI) tract. Recent preclinical findings suggest that the gut microbiome plays a pivotal role in regulating microglial maturation and function, and altered microbial community composition has been reported in neurological disorders with known microglial involvement in humans. Collectively, these findings suggest that bidirectional crosstalk between the gut and the brain may influence disease pathogenesis. Herein, we discuss recent studies showing a role for the gut microbiome in modulating microglial development and function in homeostatic and disease conditions and highlight possible future research to develop novel microbial treatments for disorders of the brain.

Emerging Developments in Microbiome and Microglia Research: Implications for Neurodevelopmental Disorders.

From immunology to neuroscience, interactions between the microbiome and host are increasingly appreciated as potent drivers of health and disease. Epidemiological studies previously identified compelling correlations between perinatal microbiome insults and neurobehavioral outcomes, the mechanistic details of which are just beginning to take shape thanks to germ-free and antibiotics-based animal models. This review summarizes parallel developments from clinical and preclinical research that suggest neuroactive roles for gut bacteria and their metabolites. We also examine the nascent field of microbiome-microglia crosstalk research, which includes pharmacological and genetic strategies to inform functional capabilities of microglia in response to microbial programming. Finally, we address an emerging hypothesis behind neurodevelopmental disorders, which implicates microbiome dysbiosis in the atypical programming of neuroimmune cells, namely microglia.

The Role of the Microbiome in the Developmental Origins of Health and Disease.

Although the prominent role of the microbiome in human health has been established, the early-life microbiome is now being recognized as a major influence on long-term human health and development. Variations in the composition and functional potential of the early-life microbiome are the result of lifestyle factors, such as mode of birth, breastfeeding, diet, and antibiotic usage. In addition, variations in the composition of the early-life microbiome have been associated with specific disease outcomes, such as asthma, obesity, and neurodevelopmental disorders. This points toward this bacterial consortium as a mediator between early lifestyle factors and health and disease. In addition, variations in the microbial intrauterine environment may predispose neonates to specific health outcomes later in life. A role of the microbiome in the Developmental Origins of Health and Disease is supported in this collective research. Highlighting the early-life critical window of susceptibility associated with microbiome development, we discuss infant microbial colonization, beginning with the maternal-to-fetal exchange of microbes in utero and up through the influence of breastfeeding in the first year of life. In addition, we review the available disease-specific evidence pointing toward the microbiome as a mechanistic mediator in the Developmental Origins of Health and Disease.

Early Childhood Neurodevelopmental Outcomes in Infants Exposed to Infectious Syphilis In Utero.

BACKGROUND: There are minimal neurodevelopmental follow-up data for infants exposed to syphilis in utero. METHODS: This is an inception cohort study of infants exposed to syphilis in utero. We reviewed women with reactive syphilis serology in pregnancy or at delivery in Edmonton (Canada), 2002 through 2010 and describe the neurodevelopmental outcomes of children with and without congenital syphilis. RESULTS: There were 39 births to women with reactive syphilis serology, 9 of whom had late latent syphilis (n = 4), stillbirths (n = 2) or early neonatal deaths (n = 3), leaving 30 survivors of which 11 with and 7 without congenital syphilis had neurodevelopmental assessment. Those with congenital syphilis were all born to women with inadequate syphilis treatment before delivery. Neurodevelopmental impairment was documented in 3 of 11 (27%) infants with congenital syphilis and one of 7 (14%) without congenital syphilis with speech language delays in 4 of 11 (36%) with congenital syphilis and 3 of 7 (42%) without congenital syphilis. CONCLUSIONS: Infants born to mothers with reactive syphilis serology during pregnancy are at high risk for neurodevelopmental impairment, whether or not they have congenital syphilis, so should all be offered neurodevelopmental assessments and early referral for services as required.

Can gut microbes play a role in mental disorders and their treatment?

The gut microbes, collectively called microbiota, are linked to the brain through a bidirectional system that involves the vagus nerve, the immune system, and various neurotransmitters. Stress response, memory functions, social behavior, and mood are modulated by microbiota. Furthermore, microbiota play a role in the development of the central nervous system. These features, established largely in rodent studies, have informed hypotheses about the role of microbiota in human psychiatric disorders. Microbiota affect phenomena that are known to be parts of the depression phenotype, such as exaggerated response to stress and inflammatory features. Furthermore, the role of microbiota in neurodevelopment and in the modulation of social behavior suggests the possibility of its role in autism spectrum disorder and in schizophrenia. If altered, microbiota play a role in psychiatric disorders, then efforts to normalize the gut microbial population by the ingestion of probiotics (live bacteria) could have antidepresssant or antipsychotic effects. Testing such hypotheses in translational human studies is a matter of future research.

The Central Nervous System and the Gut Microbiome.

Neurodevelopment is a complex process governed by both intrinsic and extrinsic signals. While historically studied by researching the brain, inputs from the periphery impact many neurological conditions. Indeed, emerging data suggest communication between the gut and the brain in anxiety, depression, cognition, and autism spectrum disorder (ASD). The development of a healthy, functional brain depends on key pre- and post-natal events that integrate environmental cues, such as molecular signals from the gut. These cues largely originate from the microbiome, the consortium of symbiotic bacteria that reside within all animals. Research over the past few years reveals that the gut microbiome plays a role in basic neurogenerative processes such as the formation of the blood-brain barrier, myelination, neurogenesis, and microglia maturation and also modulates many aspects of animal behavior. Herein, we discuss the biological intersection of neurodevelopment and the microbiome and explore the hypothesis that gut bacteria are integral contributors to development and function of the nervous system and to the balance between mental health and disease.

An observational study examining the relationship between respiratory symptoms, airway inflammation and bacteriology in children with severe neurodisability.

BACKGROUND: Children with severe neurodisability (ND) commonly suffer from chronic respiratory symptoms that impact greatly on quality of life, and lead to recurrent hospital admissions. This morbidity (and its causes) is poorly described, despite being well recognised by paediatricians. In this study, we characterised respiratory symptoms in children with ND at times of stability and deterioration. We also assessed the relationship between respiratory symptoms, lower airway inflammatory markers and levels of infection/colonisation. METHODS: ND children were recruited upon admission for elective surgery (Elective-ND [n = 16]), or acutely upon admission to Intensive Care (PICU-ND [n = 19]), and compared to healthy control children [n = 12]. Parents completed a validated respiratory symptom questionnaire in which symptoms associated with activity were removed (total maximal score of 108). Bronchoalveolar lavage (BAL) was collected, and BAL neutrophil counts, IL-8 and TGFß-1 levels measured. BAL microbial analysis was performed using a 16S/18S rRNA gene based assay and Pseudomonas aeruginosa PCR. RESULTS: All ND children had high levels of respiratory symptoms (median [IQR] symptom score PICU-ND, 55[38-64]; Elective-ND, 26[7-45]; Control, 4[0-7]: p<0.01), which affected their families, particularly at nighttime. Elective-ND patients with a total respiratory symptom score >20 invariably had BAL neutrophilia. Elective patients with 16S/18S microbial rDNA positive BAL had higher neutrophil counts (positive, 33[18-70]%; negative, 8[4-38]%: p<0.05) and generally higher symptom scores (positive, 17[5-32]; negative, 5[0-9]: p = 0.097). Streptococcus mitis was commonly identified in BAL from ND children; Pseudomonas aeruginosa was not identified in any sample. CONCLUSIONS: Children with severe ND often have high levels of chronic respiratory symptoms, which may relate to lower airway inflammation. Bacterial airway colonisation, particularly with oral commensals, may play a role in both symptom generation and inflammation.