New and Preliminary Evidence on Altered Oral and Gut Microbiota in Individuals with Autism Spectrum Disorder (ASD): Implications for ASD Diagnosis and Subtyping Based on Microbial Biomarkers.

Autism Spectrum Disorder (ASD) is a complex neurological and developmental disorder characterized by behavioral and social impairments as well as multiple co-occurring conditions, such as gastrointestinal abnormalities, dental/periodontal diseases, and allergies. The etiology of ASD likely involves interaction between genetic and environmental factors. Recent studies suggest that oral and gut microbiome play important roles in the pathogenesis of inflammation, immune dysfunction, and disruption of the gut-brain axis, which may contribute to ASD pathophysiology. The majority of previous studies used unrelated neurotypical individuals as controls, and they focused on the gut microbiome, with little attention paid to the oral flora. In this pilot study, we used a first degree-relative matched design combined with high fidelity 16S rRNA (ribosomal RNA) gene amplicon sequencing in order to characterize the oral and gut microbiotas of patients with ASD compared to neurotypical individuals, and explored the utility of microbiome markers for ASD diagnosis and subtyping of clinical comorbid conditions. Additionally, we aimed to develop microbiome biomarkers to monitor responses to a subsequent clinical trial using probiotics supplementation. We identified distinct features of gut and salivary microbiota that differed between ASD patients and neurotypical controls. We next explored the utility of some differentially enriched markers for ASD diagnosis and examined the association between the oral and gut microbiomes using network analysis. Due to the tremendous clinical heterogeneity of the ASD population, we explored the relationship between microbiome and clinical indices as an attempt to extract microbiome signatures assocociated with clinical subtypes, including allergies, abdominal pain, and abnormal dietary habits. The diagnosis of ASD currently relies on psychological testing with potentially high subjectivity. Given the emerging role that the oral and gut microbiome plays in systemic diseases, our study will provide preliminary evidence for developing microbial markers that can be used to diagnose or guide treatment of ASD and comorbid conditions. These preliminary results also serve as a starting point to test whether altering the oral and gut microbiome could improve co-morbid conditions in patients with ASD and further modify the core symptoms of ASD.

Characterization of Individual Human Antibodies That Bind Pertussis Toxin Stimulated by Acellular Immunization.

Despite high vaccination rates, the incidence of whooping cough has steadily been increasing in developing countries for several decades. The current acellular pertussis (aP) vaccines all include the major protective antigen pertussis toxin (PTx) and are safer, but they appear to be less protective than infection or older, whole-cell vaccines. To better understand the attributes of individual antibodies stimulated by aP, we isolated plasmablast clones recognizing PTx after booster immunization of two donors. Five unique antibody sequences recognizing native PTx were recovered and expressed as recombinant human IgG1 antibodies. The antibodies all bind different epitopes on the PTx S1 subunit, B oligomer, or S1-B subunit interface, and just one clone neutralized PTx in an in vitro assay. To better understand the epitopes bound by the nonneutralizing S1-subunit antibodies, comprehensive mutagenesis with yeast display provided a detailed map of the epitope recognized by antibodies A8 and E12. Residue R76 is required for antibody A8 binding and is present on the S1 surface but is only partially exposed in the holotoxin, providing a structural explanation for A8's inability to neutralize holotoxin. The B-subunit-specific antibody D8 inhibited PTx binding to a model receptor and neutralized PTx in vitro as well as in an in vivo leukocytosis assay. This is the first study, to our knowledge, to identify individual human antibodies stimulated by the acellular pertussis vaccine and demonstrates the feasibility of using these approaches to address outstanding issues in pertussis vaccinology, including mechanisms of accelerated waning of protective immunity despite repeated aP immunization.

Automated Electrophysiological and Pharmacological Evaluation of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Automated planar patch clamp systems are widely used in drug evaluation studies because of their ability to provide accurate, reliable, and reproducible data in a high-throughput manner. Typically, CHO and HEK tumorigenic cell lines overexpressing single ion channels are used since they can be harvested as high-density, homogenous, single-cell suspensions. While human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are physiologically more relevant, these cells are fragile, have complex culture requirements, are inherently heterogeneous, and are expensive to produce, which has restricted their use on automated patch clamp (APC) devices. Here, we used high efficiency differentiation protocols to produce cardiomyocytes from six different hPSC lines for analysis on the Patchliner (Nanion Technologies GmbH) APC platform. We developed a two-step cell preparation protocol that yielded cell catch rates and whole-cell breakthroughs of ∼80%, with ∼40% of these cells allowing electrical activity to be recorded. The protocol permitted formation of long-lasting (>15 min), high quality seals (>2 GΩ) in both voltage- and current-clamp modes. This enabled density of sodium, calcium, and potassium currents to be evaluated, along with dose-response curves to their respective channel inhibitors, tetrodotoxin, nifedipine, and E-4031. Thus, we show the feasibility of using the Patchliner platform for automated evaluation of the electrophysiology and pharmacology of hPSC-CMs, which will enable considerable increase in throughput for reliable and efficient drug evaluation.

Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics.

Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.

Response of corneal epithelial cells to Staphylococcus aureus.

Staphylococcus aureus is a leading cause of invasive infection. It also infects wet mucosal tissues including the cornea and conjunctiva. Conflicting evidence exists on the expression of Toll-like receptors by human corneal epithelial cells. It was therefore of interest to determine how epithelial cells from this immune privileged tissue respond to S. aureus. Further, it was of interest to determine whether cytolytic toxins, with the potential to cause ion flux or potentially permit effector molecule movement across the target cell membrane, alter the response. Microarrays were used to globally assess the response of human corneal epithelial cells to S. aureus. A large increase in abundance of transcripts encoding the antimicrobial dendritic cell chemokine, CCL20, was observed. CCL20 release into the medium was detected, and this response was found to be largely TLR2 and NOD2 independent. Corneal epithelial cells also respond to S. aureus by increasing the intracellular abundance of mRNA for inflammatory mediators, transcription factors, and genes related to MAP kinase pathways, in ways similar to other cell types. The corneal epithelial cell response was surprisingly unaffected by toxin exposure. Toxin exposure did, however, induce a stress response. Although model toxigenic and non-toxigenic strains of S. aureus were employed in the present study, the results obtained were strikingly similar to those reported for stimulation of vaginal epithelial cells by clinical toxic shock toxin expressing isolates, demonstrating that the initial epithelial cellular responses to S. aureus are largely independent of strain as well as epithelial cell tissue source.

Rapid screen for epithelial internalization of Tn917-mutagenized Streptococcus pyogenes.

Group A streptococci (GAS) cause a number of human diseases ranging from pharyngitis to necrotizing fasciitis. GAS are hypothesized to escape killing by either the immune system or beta lactam antibiotics by internalization into epithelial cells. A Tn917 library of transposon mutants was screened for capacity to invade and survive in human epithelial cells using a novel blood agar overlay method. Although the screen revealed that a majority of Tn917 insertions occurred within a 10 kb region of the genome, GAS genes identified as essential for internalization into epithelial cells included ABC transporters, and DNA maintenance proteins, and citrate metabolism enzymes, underlining the importance of adaptation to the intracellular environment.

Increased surface toll-like receptor 2 expression in superantigen shock.

OBJECTIVE: Examination of the interaction between gram-positive bacterial superantigens and toll-like receptor 2 (TLR2) in health and critical illness. DESIGN: Laboratory ex vivo model and prospective clinical, cohort study. SETTING: Two research laboratories in university hospitals and two intensive care units. SUBJECTS/PATIENTS: Laboratory study was performed in transfected HeLa cells and primary human monocytes from healthy volunteers. Clinical study used cells from 20 healthy controls and 45 critically ill patients with circulatory shock. INTERVENTIONS: HeLa cells and purified monocytes were exposed to purified superantigens or isogenic bacterial supernatants and readout obtained by cytokine enzyme-linked immunosorbent assay, flow cytometry, and quantitative real-time polymerase chain reaction. Peripheral blood mononuclear cells from patients with circulatory shock were compared with controls using flow cytometry and measurement of cytokines after ligand exposure. MEASUREMENTS AND MAIN RESULTS: Superantigens were unable to signal through ligation by TLR2. However, TLR2 was up-regulated on the surface of primary human monocytes, without detectable TLR2 messenger RNA neosynthesis, by a range of superantigens and superantigen-containing Streptococcus pyogenes supernatants, although not by isogenic superantigen-negative strains. Superantigen mutant constructs with disrupted major histocompatibility complex class II-binding sites did not support TLR2 up-regulation. TLR2 up-regulation was associated with an increase in the proinflammatory response to TLR2 ligands only at high ligand concentrations. TLR2 was up-regulated in a small subset of patients with severe S. pyogenes sepsis but not in patients with any other category of septic or circulatory shock; responses to TLR2 ligands were reduced in all categories of critically ill patient, however. CONCLUSIONS: Superantigens up-regulate monocyte surface TLR2 expression through major histocompatibility complex class II signaling. Enhanced surface TLR2 expression may be a specific feature of patients with S. pyogenes-induced shock. Importantly, intensity of TLR2 signaling is not necessarily coupled to TLR2 expression when ligand concentrations are low or after onset of critical illness.

Superantigens SPEA and SMEZ do not affect secretome expression in Streptococcus pyogenes.

The superantigens, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B (SEB), were recently reported to repress global exoprotein synthesis in Staphylococcus aureus. To investigate if this phenomenon could be observed in a different Gram-positive pathogen, the effects of two major Streptococcus pyogenes superantigens on streptococcal secretome expression were examined. Using mutagenesis and genetic complementation, we demonstrated that neither streptococcal pyrogenic exotoxin A (SPEA) nor streptococcal mitogenic exotoxin Z (SMEZ) had any consistent effect on global protein expression or on transcription of genes encoding the secreted exoproteins, DNase B, SPEB and SPEG. In S. pyogenes, superantigen production does not appear to have a major regulatory role.

C-terminal antibodies (CTAbs): a simple and broadly applicable approach for the rapid generation of protein-specific antibodies with predefined specificity.

Recent advances in proteomic techniques have resulted in an ever-increasing need to produce antibodies. Here, to address this problem, a technically simple approach of targeting the extreme C-termini of proteins with antibodies (CTAbs) was investigated in proteins secreted by the human pathogen Streptococcus pyogenes. Target proteins were identified by a conventional proteomic approach and CTAbs produced against synthetic five amino acid peptides representing the C-terminus of each target protein. In every case where protein secretion was demonstrated (n = 20), CTAbs were successfully produced and bound specifically to the target protein (100% success rate). The apparent specificity was consistent with the structural heterogeneity of the C-termini of S. pyogenes proteins. The global specificity of CTAb binding was defined using a combinatorial library of synthetic peptides representing structural variants of the original synthetic immunogen. This is a systematic and comprehensive approach for the development of antibodies with defined specificity that can be used in a range of applications.

Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes.

Lethal necrotizing fasciitis caused by Streptococcus pyogenes is characterized by a paucity of neutrophils at the site of infection. Interleukin (IL)-8, which is important for neutrophil transmigration and activation, can be degraded by S. pyogenes. Blood isolates of S. pyogenes were better able to degrade human IL-8 than throat isolates. Degradation of IL-8 was the result of a single specific cleavage between 59glutamine and 60arginine within the IL-8 C-terminal alpha helix. Cleaved IL-8 reduced neutrophil activation and migration. IL-8-cleaving activity was found in partially purified supernatant of a necrotizing fasciitis isolate, and this activity was associated with an approximately 150-kDa fraction containing S. pyogenes cell envelope proteinase (SpyCEP). IL-8-cleaving activity corresponded with the presence of SpyCEP in the supernatant. Cleavage of IL-8 by S. pyogenes represents an unprecedented mechanism of immune evasion, effectively preventing IL-8 C-terminus-mediated endothelial translocation and subsequent recruitment of neutrophils.

Superantigen recognition by HLA class II on monocytes up-regulates toll-like receptor 4 and enhances proinflammatory responses to endotoxin.

The devastating systemic effects of bacterial superantigens may be explained by powerful proinflammatory synergy with lipopolysaccharide (LPS). However, the mechanism underlying this phenomenon remains unclear and has never been investigated in humans. Specifically, there is no known link between superantigen-induced immune effects and the pattern recognition of LPS at toll-like receptor 4 (TLR4). Here we show that bacterial superantigens induce rapid transcription and increased membrane expression of TLR4 in primary human monocytes by ligation of major histocompatibility complex (MHC) class II. We also demonstrate that superantigens are solely responsible for monocyte TLR4 up-regulation induced by products from Gram-positive bacteria. In parallel with enhanced TLR4 expression, priming of purified monocytes or mixed peripheral blood mononuclear cells with superantigens significantly enhanced the induction of proinflammatory cytokines by known TLR4 ligands. Staphylococcal enterotoxin A constructs containing targeted mutations were used to demonstrate a requirement for MHC class II ligation in both TLR4 up-regulation and enhanced responses to endotoxin. In contrast to results from animal models, superantigen-endotoxin interaction was not dependent on T-cell receptor ligation by superantigen or interferon gamma production. Pattern recognition of bacterial superantigens by MHC class II receptors may exacerbate the proinflammatory response of monocytes to Gram-negative infection or endotoxin by up-regulation of TLR4.

Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans.

The use of microarrays in the analysis of gene expression is becoming widespread for many organisms, including yeast. However, although the genomes of a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms. Here, we describe the construction and validation of microarrays for the fungus Aspergillus nidulans using PCR products from a 4092 EST conidial germination library. An experiment was designed to validate these arrays by monitoring the expression profiles of known genes following the addition of 1% (w/v) glucose to wild-type A. nidulans cultures grown to mid-exponential phase in Vogel's minimal medium with ethanol as the sole carbon source. The profiles of genes showing statistically significant differential expression following the glucose up-shift are presented and an assessment of the quality and reproducibility of the A. nidulans arrays discussed.