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author:("Hornig, may")
1.  The International Collaboration for Autism Registry Epidemiology (iCARE): Multinational Registry-Based Investigations of Autism Risk Factors and Trends 
The International Collaboration for Autism Registry Epidemiology (iCARE) is the first multinational research consortium (Australia, Denmark, Finland, Israel, Norway, Sweden, USA) to promote research in autism geographical and temporal heterogeneity, phenotype, family and life course patterns, and etiology. iCARE devised solutions to challenges in multinational collaboration concerning data access security, confidentiality and management. Data are obtained by integrating existing national or state-wide, population-based, individual-level data systems and undergo rigorous harmonization and quality control processes. Analyses are performed using database federation via a computational infrastructure with a secure, web-based, interface. iCARE provides a unique, unprecedented resource in autism research that will significantly enhance the ability to detect environmental and genetic contributions to the causes and life course of autism.
doi:10.1007/s10803-013-1815-x
PMCID: PMC4512211  PMID: 23563868
Autism; Epidemiology; Study methods; Risk factors; Multinational
2.  Distinct plasma immune signatures in ME/CFS are present early in the course of illness 
Science advances  2015;1(1):e1400121.
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an unexplained incapacitating illness that may affect up to 4 million people in the United States alone. There are no validated laboratory tests for diagnosis or management despite global efforts to find biomarkers of disease. We considered the possibility that inability to identify such biomarkers reflected variations in diagnostic criteria and laboratory methods as well as the timing of sample collection during the course of the illness. Accordingly, we leveraged two large, multicenter cohort studies of ME/CFS to assess the relationship of immune signatures with diagnosis, illness duration, and other clinical variables. Controls were frequency-matched on key variables known to affect immune status, including season of sampling and geographic site, in addition to age and sex. We report here distinct alterations in plasma immune signatures early in the course of ME/CFS (n = 52) relative to healthy controls (n = 348) that are not present in subjects with longer duration of illness (n = 246). Analyses based on disease duration revealed that early ME/CFS cases had a prominent activation of both pro- and anti-inflammatory cytokines as well as dissociation of intercytokine regulatory networks. We found a stronger correlation of cytokine alterations with illness duration than with measures of illness severity, suggesting that the immunopathology of ME/CFS is not static. These findings have critical implications for discovery of interventional strategies and early diagnosis of ME/CFS.
doi:10.1126/sciadv.1400121
PMCID: PMC4465185  PMID: 26079000
3.  Identifying Children with Autism Spectrum Disorder at 18 Months in a General Population Sample 
Background
Previous research on clinical and high-risk samples suggests that signs of autism spectrum disorder (ASD) can be detected between one and two years of age. We investigated signs of ASD at 18 months in a population-based sample and the association with later ASD diagnosis.
Methods
The study sample includes 52 026 children born 2003 through 2008, and is a subset of children that participated in the Norwegian Mother and Child Cohort (MoBa), a population-based longitudinal study, and the Autism Birth Cohort (ABC), a sub-study on ASD. Parents completed all 23 items from the Modified Checklist for Autism in Toddlers (M-CHAT) at 18 months.
Results
The M-CHAT 6-critical-item criterion and the 23-item criterion had a specificity of 97.9% and 92.7% and a sensitivity of 20.8% and 34.1%, respectively. In the 173 children diagnosed with ASD to date, 60 children (34.7%) scored above the cut-off on either of the screening criteria. The items with the highest likelihood ratios were “interest in other children”, “show objects to others”, and “response to name”.
Conclusion
Even though one third of the children who later received an ASD diagnosis were identified through M-CHAT items, the majority scored below cut-off on the screening criteria at 18 months. The results imply that it might not be possible to detect all children with ASD at this age.
doi:10.1111/ppe.12114
PMCID: PMC3976700  PMID: 24547686
Autism spectrum disorders; early identification; M-CHAT; longitudinal studies; Autism Birth Cohort Study; Norwegian Mother and Child Cohort Study
4.  Parental Obesity and Risk of Autism Spectrum Disorder 
Pediatrics  2014;133(5):e1128-e1138.
OBJECTIVES:
The objective of the study was to investigate the associations among maternal prepregnancy BMI, paternal BMI, and the risk of autism spectrum disorders (ASDs) in children.
METHODS:
The study sample of 92 909 children was derived from the population-based, prospective Norwegian Mother and Child Cohort Study. The age range was 4.0 through 13.1 (mean 7.4) years. Relative risks of ASDs were estimated by odds ratios (ORs) and 95% confidence intervals (CIs) from logistic regression models.
RESULTS:
At the end of follow-up on December 31, 2012, 419 children in the study sample had been diagnosed with ASDs: 162 with autistic disorder, 103 with Asperger disorder, and 154 with pervasive developmental disorder not otherwise specified. Maternal obesity (BMI ≥30) was only weakly associated with ASD risk, whereas paternal obesity was associated with an increased risk of autistic disorder and Asperger disorder. The risk of autistic disorder was 0.27% (25 of 9267) in children of obese fathers and 0.14% (59 of 41 603) in children of fathers with normal weight (BMI <25), generating an adjusted OR of 1.73 (95% CI: 1.07–2.82). For Asperger disorder, analyses were limited to children aged ≥7 years (n = 50 116). The risk was 0.38% (18 of 4761) in children of obese fathers and 0.18% (42 of 22 736) in children of normal-weight fathers, and the adjusted OR was 2.01 (95% CI: 1.13–3.57). No associations were found for pervasive developmental disorder not otherwise specified.
CONCLUSIONS:
Paternal obesity is an independent risk factor for ASDs in children. The associations should be investigated further in genetic and epigenetic studies.
doi:10.1542/peds.2013-3664
PMCID: PMC4006442  PMID: 24709932
autism spectrum disorder; autistic disorder; Asperger disorder; PDD-NOS; parental obesity; parental BMI; child cohort study
5.  Integration of an EEG biomarker with a clinician's ADHD evaluation 
Brain and Behavior  2015;5(4):e00330.
Background
This study is the first to evaluate an assessment aid for attention-deficit/hyperactivity disorder (ADHD) according to both Class-I evidence standards of American Academy of Neurology and De Novo requirements of US Food and Drug Administration. The assessment aid involves a method to integrate an electroencephalographic (EEG) biomarker, theta/beta ratio (TBR), with a clinician's ADHD evaluation. The integration method is intended as a step to help improve certainty with criterion E (i.e., whether symptoms are better explained by another condition).
Methods
To evaluate the assessment aid, investigators conducted a prospective, triple-blinded, 13-site, clinical cohort study. Comprehensive clinical evaluation data were obtained from 275 children and adolescents presenting with attentional and behavioral concerns. A qualified clinician at each site performed differential diagnosis. EEG was collected by separate teams. The reference standard was consensus diagnosis by an independent, multidisciplinary team (psychiatrist, psychologist, and neurodevelopmental pediatrician), which is well-suited to evaluate criterion E in a complex clinical population.
Results
Of 209 patients meeting ADHD criteria per a site clinician's judgment, 93 were separately found by the multidisciplinary team to be less likely to meet criterion E, implying possible overdiagnosis by clinicians in 34% of the total clinical sample (93/275). Of those 93, 91% were also identified by EEG, showing a relatively lower TBR (85/93). Further, the integration method was in 97% agreement with the multidisciplinary team in the resolution of a clinician's uncertain cases (35/36). TBR showed statistical power specific to supporting certainty of criterion E per the multidisciplinary team (Cohen's d, 1.53). Patients with relatively lower TBR were more likely to have other conditions that could affect criterion E certainty (10 significant results; P ≤ 0.05). Integration of this information with a clinician's ADHD evaluation could help improve diagnostic accuracy from 61% to 88%.
Conclusions
The EEG-based assessment aid may help improve accuracy of ADHD diagnosis by supporting greater criterion E certainty.
doi:10.1002/brb3.330
PMCID: PMC4356845  PMID: 25798338
Attention deficit hyperactivity disorder; biomarkers; comorbidity; electroencephalography; multidisciplinary; sensitivity; specificity
6.  Towards large-cohort comparative studies to define the factors influencing the gut microbial community structure of ASD patients 
Microbial Ecology in Health and Disease  2015;26:10.3402/mehd.v26.26555.
Differences in the gut microbiota have been reported between individuals with autism spectrum disorders (ASD) and neurotypical controls, although direct evidence that changes in the microbiome contribute to causing ASD has been scarce to date. Here we summarize some considerations of experimental design that can help untangle causality in this complex system. In particular, large cross-sectional studies that can factor out important variables such as diet, prospective longitudinal studies that remove some of the influence of interpersonal variation in the microbiome (which is generally high, especially in children), and studies transferring microbial communities into germ-free mice may be especially useful. Controlling for the effects of technical variables, which have complicated efforts to combine existing studies, is critical when biological effect sizes are small. Large citizen-science studies with thousands of participants such as the American Gut Project have been effective at uncovering subtle microbiome effects in self-collected samples and with self-reported diet and behavior data, and may provide a useful complement to other types of traditionally funded and conducted studies in the case of ASD, especially in the hypothesis generation phase.
doi:10.3402/mehd.v26.26555
PMCID: PMC4355505  PMID: 25758371
autism; microbiology; microbiome; neurological
7.  Integration of an EEG biomarker with a clinician's ADHD evaluation 
Brain and Behavior  2015;e00330.
Abstract
Background
This study is the first to evaluate an assessment aid for attention‐deficit/hyperactivity disorder (ADHD) according to both Class‐I evidence standards of American Academy of Neurology and De Novo requirements of US Food and Drug Administration. The assessment aid involves a method to integrate an electroencephalographic (EEG) biomarker, theta/beta ratio (TBR), with a clinician's ADHD evaluation. The integration method is intended as a step to help improve certainty with criterion E (i.e., whether symptoms are better explained by another condition).
Methods
To evaluate the assessment aid, investigators conducted a prospective, triple‐blinded, 13‐site, clinical cohort study. Comprehensive clinical evaluation data were obtained from 275 children and adolescents presenting with attentional and behavioral concerns. A qualified clinician at each site performed differential diagnosis. EEG was collected by separate teams. The reference standard was consensus diagnosis by an independent, multidisciplinary team (psychiatrist, psychologist, and neurodevelopmental pediatrician), which is well‐suited to evaluate criterion E in a complex clinical population.
Results
Of 209 patients meeting ADHD criteria per a site clinician's judgment, 93 were separately found by the multidisciplinary team to be less likely to meet criterion E, implying possible overdiagnosis by clinicians in 34% of the total clinical sample (93/275). Of those 93, 91% were also identified by EEG, showing a relatively lower TBR (85/93). Further, the integration method was in 97% agreement with the multidisciplinary team in the resolution of a clinician's uncertain cases (35/36). TBR showed statistical power specific to supporting certainty of criterion E per the multidisciplinary team (Cohen's d, 1.53). Patients with relatively lower TBR were more likely to have other conditions that could affect criterion E certainty (10 significant results; P ≤ 0.05). Integration of this information with a clinician's ADHD evaluation could help improve diagnostic accuracy from 61% to 88%.
Conclusions
The EEG‐based assessment aid may help improve accuracy of ADHD diagnosis by supporting greater criterion E certainty.
doi:10.1002/brb3.330
PMCID: PMC4356845  PMID: 25798338
Attention deficit hyperactivity disorder; biomarkers; comorbidity; electroencephalography; multidisciplinary; sensitivity; specificity
8.  Oxidative Stress Biomarkers in Sporadic ALS 
Objective
To investigate oxidative stress biomarkers in a cross-sectional pilot study of 50 participants with sporadic ALS (sALS) compared to 46 control subjects.
Methods
We measured urinary 8-oxodeoxyguanosine (8-oxodG), urinary 15-F2t-isoprostane (IsoP), and plasma protein carbonyl by ELISA methods. We also determined if ELISA measurement of 8-oxodG could be validated against measures from high pressure liquid chromatography coupled with electrochemical detection, the current standard method.
Results
8-oxodG and IsoP levels adjusted for creatinine were significantly elevated in sALS participants. These differences persisted after age and gender were controlled in regression analyses. These markers are highly and positively correlated with each other. 8-oxodG measured by the two techniques from the same urine sample were positively correlated (P < .0001). Protein carbonyl was not different between sALS participants and controls.
Conclusion
Using ELISA we confirmed that certain oxidative stress biomarkers were elevated in sALS participants. ELISA may be reliable and thus useful in epidemiology studies requiring large numbers of samples to determine the significance of increased oxidative stress markers in sALS. Further studies are required.
doi:10.1080/17482960801933942
PMCID: PMC4332387  PMID: 18574762
epidemiology; amyotrophic lateral sclerosis (ALS); biomarkers; oxidative stress; neurodegeneration
9.  Analysis of Self-Selection Bias in a Population-Based Cohort Study of Autism Spectrum Disorders 
Paediatric and perinatal epidemiology  2013;27(6):10.1111/ppe.12077.
Background
This study examined potential self-selection bias in a large pregnancy cohort by comparing exposure-outcome associations from the cohort to similar associations obtained from nationwide registry data. The outcome under study was specialist-confirmed diagnosis of autism spectrum disorders.
Methods
The cohort sample (n = 89,836) was derived from the population-based prospective Norwegian Mother and Child Cohort Study and its sub-study of autism spectrum disorders, the Autism Birth Cohort study. The nationwide registry data were derived from the Medical Birth Registry of Norway (n = 507,856). The children were born in 1999-2007, and seven prenatal and perinatal exposures were selected for analyses.
Results
Autism spectrum disorders were reported for 234 (0.26%) children in the cohort and 2,072 (0.41%) in the nationwide population. Compared with the nationwide population, the cohort had an underrepresentation of the youngest women (<25 years), those who had single status, mothers who smoked during pregnancy, and nonusers of prenatal folic acid supplements. The ratios of the adjusted odds ratios in the cohort over the adjusted odds ratios in the nationwide population were as follows; primipara pregnancy: 1.39/1.22, prenatal folic acid use: 0.85/0.86, prenatal smoking: 1.20/1.17, preterm birth (<37 weeks): 1.48/1.42, low birthweight (<2,500 g): 1.60/1.58, male sex: 4.39/4.59 (unadjusted only); and cesarean section history: 1.03/1.04.
Conclusions
Associations estimated between autism spectrum disorders and perinatal and prenatal exposures in the cohort are close to those estimated in the nationwide population. Self-selection does not appear to compromise validity of exposure-outcome associations in the Autism Birth Cohort study.
doi:10.1111/ppe.12077
PMCID: PMC3851582  PMID: 23919580
10.  EARLY GROWTH PATTERNS IN CHILDREN WITH AUTISM 
Epidemiology (Cambridge, Mass.)  2013;24(5):660-670.
Background
Case-control studies have found increased head growth during the first year of life in children with autism spectrum disorder. Length and weight have not been as extensively studied, and there are few studies of population-based samples.
Methods
The study was conducted in a sample of 106,082 children from the population-based Norwegian Mother and Child Cohort. The children were born in 1999-2009; by the end of follow-up on 31 December 2012, the age range was 3.6 through 13.1 years (mean 7.4 years). Measures were obtained prospectively until age 12 months for head circumference and 36 months for length and weight. We compared growth trajectories in autism spectrum disorder cases and non-cases using Reed first-order models.
Results
Subjects included 376 children (310 boys and 66 girls) with specialist-confirmed autism spectrum disorder. In boys with autism spectrum disorder, mean head growth was similar to that of other boys, but variability was greater, and 8.7% had macrocephaly (head circumference>97th cohort percentile) by 12 months of age. Autism spectrum disorder boys also had slightly increased body growth, with mean length 1.1 cm above and mean weight 300 g above the cohort mean for boys at age 12 months. Throughout the first year, the head circumference of girls with autism spectrum disorder was reduced – by 0.3 cm at birth and 0.5 cm at 12 months. Their mean length was similar to that of other girls, but their mean weight was 150-350 g below at all ages from birth to three years. The reductions in mean head circumference and weight in girls with autism spectrum disorder appear to be driven by those with intellectual disability, genetic disorders and epilepsy.
Discussion
Growth trajectories in children with autism spectrum disorder diverge from those of other children and the differences are sex-specific. Previous findings of increased mean head growth were not replicated.
doi:10.1097/EDE.0b013e31829e1d45
PMCID: PMC3749377  PMID: 23867813
11.  Immune-mediated animal models of Tourette syndrome 
An autoimmune diathesis has been proposed in Tourette syndrome (TS) and related neuropsychiatric disorders such as obsessive-compulsive disorder, attention-deficit/hyperactivity disorder, autism and anorexia nervosa. Environmental triggers including infection and xenobiotics are hypothesized to lead to the production of brain-directed autoantibodies in a subset of genetically susceptible individuals. Although much work has focused on Group A Streptococcus (GAS), the role of this common childhood infection remains controversial. Animal model studies based on immune and autoantibody findings in TS have demonstrated immunoglobulin (Ig) deposits and stereotypic movements and related behavioral disturbances reminiscent of TS following exposure to GAS and other activators of host anti-microbial responses, soluble immune mediators and anti-GAS or anti-neuronal antibodies. Demonstration of the ability to recreate these abnormalities through passive transfer of serum IgG from GAS-immunized mice into naïve mice and abrogation of this activity through depletion of IgG has provided compelling evidence in support of the autoimmune hypothesis. Immunologically-based animal models of TS are a potent tool for dissecting the pathogenesis of this serious neuropsychiatric syndrome.
doi:10.1016/j.neubiorev.2013.01.007
PMCID: PMC4054816  PMID: 23313649
Tourette syndrome; obsessive-compulsive disorder; autism; Streptococcus; cytokine; autoimmunity; autoantibody; striatum; cerebellum; cytokine; stereotypic behavior; mice
12.  ASSOCIATION BETWEEN MATERNAL USE OF FOLIC ACID SUPPLEMENTS AND RISK OF AUTISM IN CHILDREN 
Context
Prenatal folic acid supplements reduce the risk of neural tube defects in children, but it has not been determined whether they protect against other neurodevelopmental disorders.
Objective
To examine the association between maternal use of prenatal folic acid supplements and the subsequent risk of autistic disorder in children.
Design, Setting, and Patients
The study sample of 85,176 was derived from the population-based, prospective Norwegian Mother and Child Cohort Study (MoBa). The children were born in 2002–08. By the end of follow-up on March 31st, 2012, the age range was 3.3–10.2 years and the mean age 6.4 years. The exposure of primary interest was use of folic acid from 4 weeks before to 8 weeks after the start of pregnancy. The start of pregnancy was defined as the first day of the last menstrual period before conception. Relative risks of ASD were estimated by odds ratios (ORs) with 95% confidence intervals (CIs) in a logistic regression analysis. Analyses were adjusted for maternal education level, year of birth, and parity.
Main Outcome Measure
Specialist-confirmed diagnosis of autistic disorder.
Results
To date, 114 children in the study sample have been diagnosed with autistic disorder. In children whose mothers took folic acid, 0.10% (64/61,042) had autistic disorder, compared with 0.21% (50/24,134) in those unexposed to folic acid. The adjusted OR for autistic disorder in children of folic acid users was 0.61 (95% CI, 0.41–0.90). Similar analyses for prenatal fish oil supplements showed no such association with autistic disorder, even though fish oil use was associated with the same maternal characteristics as folic acid use.
Conclusion
Prenatal folic acid supplements around the time of conception were associated with a lower risk of autistic disorder in the MoBa cohort.
doi:10.1001/jama.2012.155925
PMCID: PMC3908544  PMID: 23403681
13.  Absence of evidence for bornavirus infection in schizophrenia, bipolar disorder and major depressive disorder 
Molecular psychiatry  2012;17(5):486-493.
In 1983, reports of antibodies in subjects with major depressive disorder to an as-yet uncharacterized infectious agent associated with meningoencephalitis in horses and sheep led to the molecular cloning of the genome of a novel, negative-stranded neurotropic virus, Borna disease virus (BDV).1,2 This advance enabled the development of new diagnostic assays including in situ hybridization, PCR and serology based on recombinant proteins. Since these assays were first implemented in 1990 more than 80 studies have reported an association between BDV and a wide range of human illnesses that include major depressive disorder, bipolar disorder, schizophrenia, anxiety disorder, chronic fatigue syndrome, multiple sclerosis, amyotrophic lateral sclerosis, dementia and glioblastoma multiforme.3,4 However, to date there has been no blinded case-control study of the epidemiology of BDV infection. Here, in a United States-based, multi-center, yoked case-control study with standardized methods for clinical assessment and blinded serologic and molecular analysis, we report the absence of association of psychiatric illness with antibodies to BDV or with BDV nucleic acids in serially-collected serum and white blood cell samples from 396 subjects, a study population comprised of 198 matched pairs of patients and healthy controls (52 schizophrenia/control pairs, 66 bipolar disorder/control pairs, and 80 major depressive disorder/control pairs). Our results argue strongly against a role for BDV in the pathogenesis of these psychiatric disorders.
doi:10.1038/mp.2011.179
PMCID: PMC3622588  PMID: 22290118
Borna disease virus; infection; schizophrenia; affective disorders; pathogenesis
14.  A Multicenter Blinded Analysis Indicates No Association between Chronic Fatigue Syndrome/Myalgic Encephalomyelitis and either Xenotropic Murine Leukemia Virus-Related Virus or Polytropic Murine Leukemia Virus 
mBio  2012;3(5):e00266-12.
ABSTRACT
The disabling disorder known as chronic fatigue syndrome or myalgic encephalomyelitis (CFS/ME) has been linked in two independent studies to infection with xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (pMLV). Although the associations were not confirmed in subsequent studies by other investigators, patients continue to question the consensus of the scientific community in rejecting the validity of the association. Here we report blinded analysis of peripheral blood from a rigorously characterized, geographically diverse population of 147 patients with CFS/ME and 146 healthy subjects by the investigators describing the original association. This analysis reveals no evidence of either XMRV or pMLV infection.
IMPORTANCE
Chronic fatigue syndrome/myalgic encephalomyelitis has an estimated prevalence of 42/10,000 in the United States, with annual direct medical costs of $7 billion. Here, the original investigators who found XMRV and pMLV (polytropic murine leukemia virus) in blood of subjects with this disorder report that this association is not confirmed in a blinded analysis of samples from rigorously characterized subjects. The increasing frequency with which molecular methods are used for pathogen discovery poses new challenges to public health and support of science. It is imperative that strategies be developed to rapidly and coherently address discoveries so that they can be carried forward for translation to clinical medicine or abandoned to focus resource investment more productively. Our study provides a paradigm for pathogen dediscovery that may be helpful to others working in this field.
doi:10.1128/mBio.00266-12
PMCID: PMC3448165  PMID: 22991430
15.  Prenatal and Postnatal Epigenetic Programming: Implications for GI, Immune, and Neuronal Function in Autism 
Autism Research and Treatment  2012;2012:190930.
Although autism is first and foremost a disorder of the central nervous system, comorbid dysfunction of the gastrointestinal (GI) and immune systems is common, suggesting that all three systems may be affected by common molecular mechanisms. Substantial systemic deficits in the antioxidant glutathione and its precursor, cysteine, have been documented in autism in association with oxidative stress and impaired methylation. DNA and histone methylation provide epigenetic regulation of gene expression during prenatal and postnatal development. Prenatal epigenetic programming (PrEP) can be affected by the maternal metabolic and nutritional environment, whereas postnatal epigenetic programming (PEP) importantly depends upon nutritional support provided through the GI tract. Cysteine absorption from the GI tract is a crucial determinant of antioxidant capacity, and systemic deficits of glutathione and cysteine in autism are likely to reflect impaired cysteine absorption. Excitatory amino acid transporter 3 (EAAT3) provides cysteine uptake for GI epithelial, neuronal, and immune cells, and its activity is decreased during oxidative stress. Based upon these observations, we propose that neurodevelopmental, GI, and immune aspects of autism each reflect manifestations of inadequate antioxidant capacity, secondary to impaired cysteine uptake by the GI tract. Genetic and environmental factors that adversely affect antioxidant capacity can disrupt PrEP and/or PEP, increasing vulnerability to autism.
doi:10.1155/2012/190930
PMCID: PMC3420412  PMID: 22934169
16.  Application of Novel PCR-Based Methods for Detection, Quantitation, and Phylogenetic Characterization of Sutterella Species in Intestinal Biopsy Samples from Children with Autism and Gastrointestinal Disturbances 
mBio  2012;3(1):e00261-11.
ABSTRACT
Gastrointestinal disturbances are commonly reported in children with autism and may be associated with compositional changes in intestinal bacteria. In a previous report, we surveyed intestinal microbiota in ileal and cecal biopsy samples from children with autism and gastrointestinal dysfunction (AUT-GI) and children with only gastrointestinal dysfunction (Control-GI). Our results demonstrated the presence of members of the family Alcaligenaceae in some AUT-GI children, while no Control-GI children had Alcaligenaceae sequences. Here we demonstrate that increased levels of Alcaligenaceae in intestinal biopsy samples from AUT-GI children result from the presence of high levels of members of the genus Sutterella. We also report the first Sutterella-specific PCR assays for detecting, quantitating, and genotyping Sutterella species in biological and environmental samples. Sutterella 16S rRNA gene sequences were found in 12 of 23 AUT-GI children but in none of 9 Control-GI children. Phylogenetic analysis revealed a predominance of either Sutterella wadsworthensis or Sutterella stercoricanis in 11 of the individual Sutterella-positive AUT-GI patients; in one AUT-GI patient, Sutterella sequences were obtained that could not be given a species-level classification based on the 16S rRNA gene sequences of known Sutterella isolates. Western immunoblots revealed plasma IgG or IgM antibody reactivity to Sutterella wadsworthensis antigens in 11 AUT-GI patients, 8 of whom were also PCR positive, indicating the presence of an immune response to Sutterella in some children.
IMPORTANCE
Autism spectrum disorders affect ~1% of the population. Many children with autism have gastrointestinal (GI) disturbances that can complicate clinical management and contribute to behavioral problems. Understanding the molecular and microbial underpinnings of these GI issues is of paramount importance for elucidating pathogenesis, rendering diagnosis, and administering informed treatment. Here we describe an association between high levels of intestinal, mucoepithelial-associated Sutterella species and GI disturbances in children with autism. These findings elevate this little-recognized bacterium to the forefront by demonstrating that Sutterella is a major component of the microbiota in over half of children with autism and gastrointestinal dysfunction (AUT-GI) and is absent in children with only gastrointestinal dysfunction (Control-GI) evaluated in this study. Furthermore, these findings bring into question the role Sutterella plays in the human microbiota in health and disease. With the Sutterella-specific molecular assays described here, some of these questions can begin to be addressed.
doi:10.1128/mBio.00261-11
PMCID: PMC3252763  PMID: 22233678
17.  Toll-like Receptor 3 Regulates Neural Stem Cell Proliferation by Modulating the Sonic Hedgehog Pathway 
PLoS ONE  2011;6(10):e26766.
Toll-like receptor 3 (TLR3) signaling has been implicated in neural stem/precursor cell (NPC) proliferation. However, the molecular mechanisms involved, and their relationship to classical TLR-mediated innate immune pathways, remain unknown. Here, we report investigation of the mechanics of TLR3 signaling in neurospheres comprised of epidermal growth factor (EGF)-responsive NPC isolated from murine embryonic cerebral cortex of C57BL/6 (WT) or TLR3 deficient (TLR3−/−) mice. Our data indicate that the TLR3 ligand polyinosinic-polycytidylic acid (PIC) negatively regulates NPC proliferation by inhibiting Sonic Hedgehog (Shh) signaling, that PIC induces apoptosis in association with inhibition of Ras-ERK signaling and elevated expression of Fas, and that these effects are TLR3-dependent, suggesting convergent signaling between the Shh and TLR3 pathways.
doi:10.1371/journal.pone.0026766
PMCID: PMC3201973  PMID: 22046349
18.  Impaired Carbohydrate Digestion and Transport and Mucosal Dysbiosis in the Intestines of Children with Autism and Gastrointestinal Disturbances 
PLoS ONE  2011;6(9):e24585.
Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism.
doi:10.1371/journal.pone.0024585
PMCID: PMC3174969  PMID: 21949732
20.  Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection 
Although scarce after annual influenza vaccination, B cells producing antibodies capable of neutralizing multiple influenza strains are abundant in humans infected with pandemic 2009 H1N1 influenza.
The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.
doi:10.1084/jem.20101352
PMCID: PMC3023136  PMID: 21220454
21.  Bocavirus Episome in Infected Human Tissue Contains Non-Identical Termini 
PLoS ONE  2011;6(6):e21362.
Human bocaviruses (HBoV) are highly prevalent human infections whose pathogenic potential remains unknown. Recent identification of the first non-human primate bocavirus [1] in captive gorillas raised the possibility of the persistent nature of bocavirus infection. To characterize bocavirus infection in humans, we tested intestinal biopsies from 22 children with gastrointestinal disease for the presence of HBoV DNA. Four HBoV-positive tissue samples were analyzed to determine whether viral DNA was present in the linear genomic, the episomal closed circular or the host genome-integrated form. Whereas one tissue sample positive for HBoV3 contained the episomal form (HBoV3-E1), none had the genome-integrated form. The complete genome sequence of HBoV3-E1 contains 5319 nucleotides of which 513 represent the non-coding terminal sequence. The secondary structure of HBoV3-E1 termini suggests several conserved and variable features among human and animal bocaviruses. Our observation that HBoV genome exists as head-to-tail monomer in infected tissue either reflects the likely evolution of alternative replication mechanism in primate bocaviruses or a mechanism of viral persistence in their host. Moreover, we identified the HBoV genomic terminal sequences that will be helpful in developing reverse genetic systems for these widely prevalent parvoviruses.
Significance
HBoV have been found in healthy human controls as well as individuals with respiratory or gastrointestinal disease. Our findings suggest that HBoV DNA can exist as episomes in infected human tissues and therefore can likely establish persistent infection in the host. Previous efforts to grow HBoV in cell culture and to develop reverse genetic systems have been unsuccessful. Complete genomic sequence of the HBoV3 episome and its genomic termini will improve our understanding of HBoV replication mechanism and its pathogenesis.
doi:10.1371/journal.pone.0021362
PMCID: PMC3125170  PMID: 21738642
22.  THE AUTISM BIRTH COHORT (ABC): A PARADIGM FOR GENE-ENVIRONMENT-TIMING RESEARCH 
Molecular psychiatry  2010;15(7):676-680.
The reported prevalence of autism spectrum disorders (ASD) has increased 5–10× over the past 20 years. Whether ASD are truly more frequent is controversial; nonetheless, the burden is profound in human and economic terms. Although autism is among the most heritable of mental disorders, its pathogenesis remains obscure. Environmental factors are proposed; however, none is implicated. Furthermore, there are no biomarkers to screen for ASD or risk of ASD. The Autism Birth Cohort (ABC) was initiated to investigate gene × environment × timing interactions and enable early diagnosis. It employs a large, unselected birth cohort wherein cases are prospectively ascertained through population screening. Samples collected serially through pregnancy and childhood include parental blood, maternal urine, cord blood, milk teeth and rectal swabs. More than 107 000 children are continuously screened via questionnaires, referral and a national registry. Cases are compared with a control group from the same cohort in a “nested case-control” design. Early screening, diagnostic assessments and re-assessments are designed to provide a rich view of longitudinal trajectory. Genetic, proteomic, immunologic, metagenomic and microbiological tools will be used to exploit unique biological samples. The ABC is a paradigm for investigating the role of genetic and environmental factors in complex disorders.
doi:10.1038/mp.2009.143
PMCID: PMC2892398  PMID: 20571529
Autism; neurodevelopmental disorder; birth cohort; biobank; molecular biology; genes and environment
23.  Induction of Toll-Like Receptor 3-Mediated Immunity during Gestation Inhibits Cortical Neurogenesis and Causes Behavioral Disturbances 
mBio  2010;1(4):e00176-10.
Maternal infection during pregnancy with a wide range of RNA and DNA viruses is associated with increased risk for schizophrenia and autism in their offspring. A common feature in these exposures is that virus replication induces innate immunity through interaction with Toll-like receptors (TLRs). We employed a mouse model wherein pregnant mice were exposed to polyinosinic-polycytidylic acid [poly(I  ⋅  C)], a synthetic, double-stranded RNA molecular mimic of replicating virus. Poly(I ⋅ C) inhibited embryonic neuronal stem cell replication and population of the superficial layers of the neocortex by neurons. Poly(I ⋅ C) also led to impaired neonatal locomotor development and abnormal sensorimotor gating responses in adult offspring. Using Toll-like receptor 3 (TLR3)-deficient mice, we established that these effects were dependent on TLR3. Inhibition of stem cell proliferation was also abrogated by pretreatment with the nonsteroidal anti-inflammatory drug (NSAID) carprofen, a cyclooxygenase (COX) inhibitor. Our findings provide insights into mechanisms by which maternal infection can induce subtle neuropathology and behavioral dysfunction, and they may suggest strategies for reducing the risk of neuropsychiatric disorders subsequent to prenatal exposures to pathogens and other triggers of innate immunity.
IMPORTANCE
Maternal infection during gestation increases the risk of neuropsychiatric disorders in their offspring. Furthermore, work in animal models indicates that pre- or neonatal infections with a wide range of viruses results in similar neurodevelopmental outcomes. These observations are consistent with a mechanism whereby damage is mediated through common pathways. Exposure of pregnant mice to polyinosinic-polycytidylic acid [poly(I ⋅ C)], a synthetic, double-stranded RNA (dsRNA) molecular mimic of replicating virus, inhibited embryonic neuronal stem cell replication and led to behavioral abnormalities in their offspring. These effects were mediated through TLR3 and abrogated by pretreatment with the nonsteroidal anti-inflammatory drug (NSAID) carprofen. Our findings provide insights into mechanisms by which maternal infection can induce subtle neuropathology and may suggest strategies for reducing the risk of neuropsychiatric diseases following exposures to infectious agents and other triggers of innate immunity during gestation.
doi:10.1128/mBio.00176-10
PMCID: PMC2953007  PMID: 20941330
24.  Heart and Skeletal Muscle Inflammation of Farmed Salmon Is Associated with Infection with a Novel Reovirus 
PLoS ONE  2010;5(7):e11487.
Atlantic salmon (Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999[1], HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom[2]. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Koch's postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations.
doi:10.1371/journal.pone.0011487
PMCID: PMC2901333  PMID: 20634888
25.  Nonparametric methods for the analysis of single-color pathogen microarrays 
BMC Bioinformatics  2010;11:354.
Background
The analysis of oligonucleotide microarray data in pathogen surveillance and discovery is a challenging task. Target template concentration, nucleic acid integrity, and host nucleic acid composition can each have a profound effect on signal distribution. Exploratory analysis of fluorescent signal distribution in clinical samples has revealed deviations from normality, suggesting that distribution-free approaches should be applied.
Results
Positive predictive value and false positive rates were examined to assess the utility of three well-established nonparametric methods for the analysis of viral array hybridization data: (1) Mann-Whitney U, (2) the Spearman correlation coefficient and (3) the chi-square test. Of the three tests, the chi-square proved most useful.
Conclusions
The acceptance of microarray use for routine clinical diagnostics will require that the technology be accompanied by simple yet reliable analytic methods. We report that our implementation of the chi-square test yielded a combination of low false positive rates and a high degree of predictive accuracy.
doi:10.1186/1471-2105-11-354
PMCID: PMC2909221  PMID: 20584331

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