Precision synbiotics increase gut microbiome diversity and improve gastrointestinal symptoms in a pilot open-label study for autism spectrum disorder.

The efficacy of prebiotics and probiotics (synbiotics when combined) to improve symptoms associated with autism spectrum disorder (ASD) has shown considerable inter-study variation, likely due to the complex, heterogeneous nature of the disorder and its associated behavioral, developmental, and gastrointestinal symptoms. Here, we present a precision synbiotic supplementation study in 296 children and adults diagnosed with ASD versus 123 age-matched neurotypical controls. One hundred seventy ASD participants completed the study. Baseline and post-synbiotic assessment of ASD and gastrointestinal (GI) symptoms and deep metagenomic sequencing were performed. Within the ASD cohort, there were significant differences in microbes between subpopulations based on the social responsiveness scale (SRS2) survey (Prevotella spp., Bacteroides, Fusicatenibacter, and others) and gluten and dairy-free diets (Bifidobacterium spp., Lactococcus, Streptococcus spp., and others). At the baseline, the ASD cohort maintained a lower taxonomic alpha diversity and significant differences in taxonomic composition, metabolic pathways, and gene families, with a greater proportion of potential pathogens, including Shigella, Klebsiella, and Clostridium, and lower proportions of beneficial microbes, including Faecalibacterium compared to controls. Following the 3-month synbiotic supplementation, the ASD cohort showed increased taxonomic alpha diversity, shifts in taxonomy and metabolic pathway potential, and improvements in some ASD-related symptoms, including a significant reduction in GI discomfort and overall improved language, comprehension, cognition, thinking, and speech. However, the open-label study design may include some placebo effects. In summary, we found that precision synbiotics modulated the gut microbiome and could be used as supplementation to improve gastrointestinal and ASD-related symptoms. IMPORTANCE: Autism spectrum disorder (ASD) is prevalent in 1 out of 36 children in the United States and contributes to health, financial, and psychological burdens. Attempts to identify a gut microbiome signature of ASD have produced varied results. The limited pre-clinical and clinical population sizes have hampered the success of these trials. To understand the microbiome associated with ASD, we employed whole metagenomic shotgun sequencing to classify microbial composition and genetic functional potential. Despite being one of the most extensive ASD post-synbiotic assessment studies, the results highlight the complexity of performing such a case-control supplementation study in this population and the potential for a future therapeutic approach in ASD.

Sensitive GC-MS/MS method to measure deuterium labeled deoxyadenosine in DNA from limited mouse cell populations.

A rapid and sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to quantitatively measure low levels of DNA base deoxyadenosine (dA) and its isotopologues (e.g., dA M+1) from limited mouse cell populations. Mice undergoing allogeneic hematopoietic transplantation (AHSCT) received deuterated water at biologically relevant time intervals post AHSCT, allowing labeling of DNA upon cell division, which was detected as the dA M+1 isotopologue. Targeted mouse cell populations were isolated from lymphoid organs and purified by multiparameter fluorescence activated cell sorting. Cell lysis, DNA extraction, and hydrolysis were accomplished using available commercial procedures. The novel analytical method utilized a hydrophilic-lipophilic balanced sample preparation, rapid online hot GC inlet gas phase sample derivatization, fast GC low thermal mass technology, and a recently marketed GC-MS/MS system. Calibration standards containing dA and fortified with relevant levels of dA M+1 (0.25-20%) and dA M+5 (internal standard) were used for sample quantitation. The method employed a quadratic fit for calibration of dA M+1 (0.25-20%) and dA, demonstrated excellent accuracy and precision, and had limits of detection of 100 fg on-column for the dA isotopologues. The method was validated and required only 20 000 cells to characterize population dynamics of cells involved in the biology of chronic graft-versus-host disease, the main cause of late morbidity and nonrelapse-mortality following AHSCT. The high sensitivity and specificity of the method makes it useful for investigating in vivo kinetics on limited and important cell populations (e.g., T regulatory cells) from disease conditions or in disease models that are immune-mediated, such as diabetes, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), arthritis, inflammatory bowel disease, and multiple sclerosis.

Pentostatin plus cyclophosphamide safely and effectively prevents immunotoxin immunogenicity in murine hosts.

PURPOSE: The success of immunotoxin therapy of cancer is limited by host production of neutralizing antibodies, which are directed toward the Pseudomonas exotoxin A (PE) component. In this proof-of-principle study using a well-established murine model, we hypothesized that a newly developed immune depletion regimen consisting of pentostatin plus cyclophosphamide would abrogate anti-immunotoxin reactivity. EXPERIMENTAL DESIGN: BALB/c hosts were injected weekly with recombinant immunotoxin (RIT) SS1P, which is an antimesothelin Fv antibody fragment genetically fused to a 38 kDa portion of PE, and has been evaluated in clinical trials. Experimental cohorts received induction chemotherapy consisting of pentostatin (P) plus cyclophosphamide (C) prior to initial RIT exposure; some cohorts received further maintenance PC therapy of varying intensity just prior to each weekly RIT challenge. Cohorts were monitored for T, B, myeloid cell depletion, and for total anti-SS1P antibody (Ab) formation. RESULTS: Controls uniformly developed anti-SS1P Ab after the third RIT exposure. Induction PC therapy reduced the frequency of hosts with anti-SS1P Ab. Abrogation of antibody generation was improved by maintenance PC therapy: nearly 100% of recipients of intensive PC maintenance were free of anti-SS1P Ab after 9 weekly RIT doses. The most effective PC regimen yielded the greatest degree of host B-cell depletion, moderate T-cell depletion, and minimal myeloid cell depletion. CONCLUSIONS: Induction and maintenance PC chemotherapy safely prevented anti-immunotoxin antibody formation with uniform efficacy. These data suggest that immunotoxin therapy might be used in combination with pentostatin plus cyclophosphamide chemotherapy to improve the targeted therapy of cancer.