Brent L. Williams

Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances.

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.

Novel Borna Virus in Psittacine Birds with Proventricular Dilatation Disease

Pyrosequencing of cDNA from brains of parrots with proventricular dilatation disease (PDD), an unexplained fatal inflammatory central, autonomic, and peripheral nervous system disease, showed 2 strains of a novel Borna virus. Real-time PCR confirmed virus presence in brain, proventriculus, and adrenal gland of 3 birds with PDD but not in 4 unaffected birds.

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

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. 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.

Genital inflammation and the risk of HIV acquisition in women

BACKGROUND Women in Africa, especially young women, have very high human immunodeficiency virus (HIV) incidence rates that cannot be fully explained by behavioral risks. We investigated whether genital inflammation influenced HIV acquisition in this group. METHODS Twelve selected cytokines, including 9 inflammatory cytokines and chemokines (interleukin [IL]-1α, IL-1β, IL-6, tumor necrosis factor-α, IL-8, interferon-γ inducible protein-10 [IP-10], monocyte chemoattractant protein-1, macrophage inflammatory protein [MIP]-1α, MIP-1β), hematopoietic IL-7, and granulocyte macrophage colony-stimulating factor, and regulatory IL-10 were measured prior to HIV infection in cervicovaginal lavages from 58 HIV seroconverters and 58 matched uninfected controls and in plasma from a subset of 107 of these women from the Centre for the AIDS Programme of Research in South Africa 004 tenofovir gel trial. RESULTS HIV seroconversion was associated with raised genital inflammatory cytokines (including chemokines MIP-1α, MIP-1β, and IP-10). The risk of HIV acquisition was significantly higher in women with evidence of genital inflammation, defined by at least 5 of 9 inflammatory cytokines being raised (odds ratio, 3.2; 95% confidence interval, 1.3-7.9; P = .014). Genital cytokine concentrations were persistently raised (for about 1 year before infection), with no readily identifiable cause despite extensive investigation of several potential factors, including sexually transmitted infections and systemic cytokines. CONCLUSIONS Elevated genital concentrations of HIV target cell-recruiting chemokines and a genital inflammatory profile contributes to the high risk of HIV acquisition in these African women.

NS1 Protein Secretion during the Acute Phase of West Nile Virus Infection

ABSTRACT The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

Bocavirus Episome in Infected Human Tissue Contains Non-Identical Termini

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.

Tropospheric winds from northeastern China carry the etiologic agent of Kawasaki disease from its source to Japan

Significance Kawasaki disease (KD), the leading cause of acquired heart disease in children worldwide, has remained a mystery for more than 40 y. No etiological agent has yet been identified. By using simulations with the flexible particle dispersion model from different Japanese cities from each single high (low) KD incidence day, the source region KD is retrieved in cereal croplands in northeastern China. We infer the incubation time for KD ranges from 6 h to 2 d, thus favoring an antigenic or toxic exposure as the trigger. Candida sp. is reported as the dominant fungal species collected aloft (54% of all fungal DNA clones) demonstrating the potential for human disease in aerosols transported by wind currents traveling long distances. Evidence indicates that the densely cultivated region of northeastern China acts as a source for the wind-borne agent of Kawasaki disease (KD). KD is an acute, coronary artery vasculitis of young children, and still a medical mystery after more than 40 y. We used residence times from simulations with the flexible particle dispersion model to pinpoint the source region for KD. Simulations were generated from locations spanning Japan from days with either high or low KD incidence. The postepidemic interval (1987–2010) and the extreme epidemics (1979, 1982, and 1986) pointed to the same source region. Results suggest a very short incubation period (<24 h) from exposure, thus making an infectious agent unlikely. Sampling campaigns over Japan during the KD season detected major differences in the microbiota of the tropospheric aerosols compared with ground aerosols, with the unexpected finding of the Candida species as the dominant fungus from aloft samples (54% of all fungal strains). These results, consistent with the Candida animal model for KD, provide support for the concept and feasibility of a windborne pathogen. A fungal toxin could be pursued as a possible etiologic agent of KD, consistent with an agricultural source, a short incubation time and synchronized outbreaks. Our study suggests that the causative agent of KD is a preformed toxin or environmental agent rather than an organism requiring replication. We propose a new paradigm whereby an idiosyncratic immune response, influenced by host genetics triggered by an environmental exposure carried on winds, results in the clinical syndrome known as acute KD.

Hippocampal Poly(ADP-Ribose) Polymerase 1 and Caspase 3 Activation in Neonatal Bornavirus Infection

ABSTRACT Infection of neonatal rats with Borna disease virus results in a characteristic behavioral syndrome and apoptosis of subsets of neurons in the hippocampus, cerebellum, and cortex (neonatal Borna disease [NBD]). In the NBD rat hippocampus, dentate gyrus granule cells progressively degenerate. Apoptotic loss of granule cells in NBD is associated with accumulation of zinc in degenerating neurons and reduced zinc in granule cell mossy fibers. Excess zinc can trigger poly(ADP-ribose) polymerase 1 (PARP-1) activation, and PARP-1 activation can mediate neuronal death. Here, we evaluate hippocampal PARP-1 mRNA and protein expression levels, activation, and cleavage, as well as apoptosis-inducing factor (AIF) nuclear translocation and executioner caspase 3 activation, in NBD rats. PARP-1 mRNA and protein levels were increased in NBD hippocampi. PARP-1 expression and activity were increased in granule cell neurons and glia with enhanced ribosylation of proteins, including PARP-1 itself. In contrast, levels of poly(ADP-ribose) glycohydrolase mRNA were decreased in NBD hippocampi. PARP-1 cleavage and AIF expression were also increased in astrocytes in NBD hippocampi. Levels of activated caspase 3 protein were increased in NBD hippocampi and localized to nuclei, mossy fibers, and dendrites of granule cell neurons. These results implicate aberrant zinc homeostasis, PARP-1, and caspase 3 activation as contributing factors in hippocampal neurodegeneration in NBD.

Metallothioneins and Zinc Dysregulation Contribute to Neurodevelopmental Damage in a Model of Perinatal Viral Infection

Neonatal Borna disease (NBD) virus infection in the Lewis rat results in life‐long viral persistence and causes behavioral and neurodevelopmental abnormalities. A hallmark of the disorder is progressive loss of cerebellar Purkinje and dentate gyrus granule cells. Findings of increased brain metallothionein‐I and‐II (MT‐I/‐II) mRNA expression in cDNA microarray experiments led us to investigate MT isoforms and their relationship to brain zinc metabolism, cellular toxicity, and neurodevelopmental abnormalities in this model. Real‐time PCR confirmed marked induction of MT‐I/‐II mRNA expression in the brains of NBD rats (40.5‐fold increase in cerebellum, p<0.0001; 6.8‐fold increase in hippocampus, p = 0.003; and 9.5‐fold increase in striatum, p = 0.0012), whereas a trend toward decreased MT‐III mRNA was found in hippocampus (1.25‐fold decrease, p = 0.0841). Double label immunofluorescence revealed prominent MT‐I/‐II expression in astrocytes throughout the brain; MT‐III protein was decreased in granule cell neurons and increased in astrocytes, with differential subcellular distribution from cytoplasmic to nuclear compartments in NBD rat hippocampus. Modified Timm staining of hippocampus revealed reduced zinc in mossy fiber projections to the hilus and CA3, accumulation of zinc in glial cells and degenerating granule cell somata, and robust mossy fiber sprouting into the inner molecular layer of the dentate gyrus. Zinc Transporter 3 (ZnT‐3) mRNA expression was decreased in hippocampus (2.3‐fold decrease, p = 0.0065); staining for its correlate protein was reduced in hippocampal mossy fibers. Furthermore, 2 molecules implicated in axonal pathfinding and mossy fiber sprouting, the extracellular matrix glycoprotein, tenascin‐R (TN‐R), and the hyaluronan receptor CD44, were increased in NBD hippocampal neuropil. Abnormal zinc metabolism and mechanisms of neuroplasticity may contribute to the pathogenesis of disease in this model, raising more general implications for neurodevelopmental damage following viral infections in early life.

Maternal Antibiotic Treatment Impacts Development of the Neonatal Intestinal Microbiome and Antiviral Immunity

Microbial colonization of the infant gastrointestinal tract (GIT) begins at birth, is shaped by the maternal microbiota, and is profoundly altered by antibiotic treatment. Antibiotic treatment of mothers during pregnancy influences colonization of the GIT microbiota of their infants. The role of the GIT microbiota in regulating adaptive immune function against systemic viral infections during infancy remains undefined. We used a mouse model of perinatal antibiotic exposure to examine the effect of GIT microbial dysbiosis on infant CD8+ T cell–mediated antiviral immunity. Maternal antibiotic treatment/treated (MAT) during pregnancy and lactation resulted in profound alterations in the composition of the GIT microbiota in mothers and infants. Streptococcus spp. dominated the GIT microbiota of MAT mothers, whereas Enterococcus faecalis predominated within the MAT infant GIT. MAT infant mice subsequently exhibited increased and accelerated mortality following vaccinia virus infection. Ag-specific IFN-γ–producing CD8+ T cells were reduced in sublethally infected MAT infant mice. MAT CD8+ T cells from uninfected infant mice also demonstrated a reduced capacity to sustain IFN-γ production following in vitro activation. We additionally determined that control infant mice became more susceptible to infection if they were born in an animal facility using stricter standards of hygiene. These data indicate that undisturbed colonization and progression of the GIT microbiota during infancy are necessary to promote robust adaptive antiviral immune responses.