Adenosine and Autism - Recent Research and a New Perspective

Autism Spectrum Disorders (ASD) are associated with atypical social, behavioral and physiological characteristics. Here we outline an emerging connection among the increased incidence of epilepsy, disrupted sleep and perseverative behaviors exhibited and sought by persons with autism. Specifically, we propose that persons with autism can benefit from increased levels of adenosine, a powerful inhibitory neuromodulator and the core molecule of adenosine triphosphate (ATP). We review the literature and present recent data obtained via a customized questionnaire administered to parents of children with a confirmed autism diagnosis. This customized questionnaire demonstrates that symptoms of autism are reduced subsequent to stimuli predicted to increase adenosine. In addition, we present evidence from the literature and pilot data from a retrospective study of children with epilepsy or epilepsy and autistic behavior who were treated with a ketogenic diet, a long established anticonvulsant therapy that recently has been shown to suppress seizures via the adenosine A1 receptor (A1R) subtype. Our discussion focuses on the actions of adenosine in the central nervous system, with multiple implications for ASD, and the potential for developing new evidence-based therapies. Taken together, published peer-reviewed research and recent preliminary research suggest that adenosine could help resolve multiple physiological and behavioral symptoms of ASD

Ketogenic Diet Improves Core Symptoms of Autism in BTBR Mice

Autism spectrum disorders share three core symptoms: impaired sociability, repetitive behaviors and communication deficits. Incidence is rising, and current treatments are inadequate. Seizures are a common comorbidity, and since the 1920’s a high-fat, low-carbohydrate ketogenic diet has been used to treat epilepsy. Evidence suggests the ketogenic diet and analogous metabolic approaches may benefit diverse neurological disorders. Here we show that a ketogenic diet improves autistic behaviors in the BTBR mouse. Juvenile BTBR mice were fed standard or ketogenic diet for three weeks and tested for sociability, self-directed repetitive behavior, and communication. In separate experiments, spontaneous intrahippocampal EEGs and tests of seizure susceptibility (6 Hz corneal stimulation, flurothyl, SKF83822, pentylenetetrazole) were compared between BTBR and control (C57Bl/6) mice. Ketogenic diet-fed BTBR mice showed increased sociability in a three-chamber test, decreased self-directed repetitive behavior, and improved social communication of a food preference. Although seizures are a common comorbidity with autism, BTBR mice fed a standard diet exhibit neither spontaneous seizures nor abnormal EEG, and have increased seizure susceptibility in just one of four tests. Thus, behavioral improvements are dissociable from any antiseizure effect. Our results suggest that a ketogenic diet improves multiple autistic behaviors in the BTBR mouse model. Therefore, ketogenic diets or analogous metabolic strategies may offer novel opportunities to improve core behavioral symptoms of autism spectrum disorders

Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation.

Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. The localization of these factors, their bioactivity and natural countermeasures remain unclear. This concept was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. The Inflammatory and inhibitory potentials of the identified proteins were measured using T cells assays. A potent pro-inflammatory factor was detected in BALF, and we hypothesized SEA could be recovered with its biological activity. Highly purified BALF fractions with bioactivity were subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. Inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show a fraction of bioactive SEA resided in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail

TNF and CD28 Signaling Play Unique but Complementary Roles in the Systemic Recruitment of Innate Immune Cells after Staphylococcus aureus Enterotoxin A Inhalation

TNF and CD28 Signaling Play Unique but Complementary Roles in the Systemic Recruitment of Innate Immune Cells after Staphylococcus aureus Enterotoxin A Inhalation

SEA induces classical pro-inflammatory factors in BALF.

<p>BALF obtained 2–88 h after i.n. SEA inhalation was tested for the presence of IL-6, TNF and SAA by ELISA. Plots show cytokine secretion, each symbol represents one mouse. BALF were collected from 10 experiments. The error bars indicate the standard error of the mean between the mice in one group. Statistical significance between the groups was evaluated by two-tailed Student’s <i>t</i> tests.</p

Overnight incubation with lactoferrin affects cell viability.

<p>Cell were treated with proteins at 5 mg/ml, except Exp. #4* (10 mg/ml), stimulated for 18 h as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g006" target="_blank">Fig 6a and 6b</a>. Viability of cells was then assayed by flow cytometry using Mitoflow as a marker of mitochondrial integrity, the percentage of cell alive is reported in the table.</p><p>Overnight incubation with lactoferrin affects cell viability.</p

3 h incubation with lactoferrin does not affect cell viability.

<p>Cell were treated with proteins (5 mg/ml), washed as described as in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g006" target="_blank">Fig 6c</a>, and stimulated for 18 h as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141548#pone.0141548.g006" target="_blank">Fig 6c and 6e</a>. Viability of cells was then assessed by flow cytometry using Mitoflow as a measurement of mitochondrial integrity, the percentage of cells alive is reported in the table.</p><p>3 h incubation with lactoferrin does not affect cell viability.</p

Lactoferrin co-migrates with SEA.

<p>(<b>A</b>) BALF-SEA was resolved by 4–15% gradient SDS-PAGE under reducing and denaturing conditions, transferred to nitrocellulose membrane and probed with anti-lactoferrin and anti-SEA antibodies as described in the Materials and Methods. Purified Lactoferrin and SEA (30 and 10 ng) were loaded in parallel to serve as positive controls. Specificity of the antibody was assessed using isotype control. The data are representative of 2 (lactoferrin) and 3 (SEA) independent experiments. (<b>B</b>) 16 h BALF-SEA was directly fractionated by cation exchange chromatography. Fractions were tested in a bioassay and immunoblotted for lactoferrin (inset). Chromatogram of protein absorption at 280nM (top panel) and bar graphs of IL-2 secretion (bottom panel) are shown. Representative of 1 out 3 experiments is shown. (<b>C</b>) BALF from naïve mice, and BALF from mice having received a time course of i.n. SEA and BSS were immunoblotted with anti-lactoferrin antibodies and 3 experiments are shown.</p