A comparative study on dietary diversity and gut microbial diversity in children with autism spectrum disorder, attention-deficit hyperactivity disorder, their neurotypical siblings, and non-related neurotypical volunteers: a cross-sectional study.

BACKGROUND: Previous research has shown a significant link between gut microbiota in children with autism spectrum disorder (ASD) and attention-deficit hyperactivity disorder (ADHD). However, much remains unknown because of the heterogeneity of disorders and the potential confounders such as dietary patterns and control group variations. METHODS: Children aged 6-12 years who had been clinically diagnosed with ASD and/or ADHD, their unaffected neurotypical siblings, and non-related neurotypical volunteers were recruited cross-sectionally. The ASD diagnosis was confirmed using the Autism Diagnostic Observation Schedule-2 (ADOS-2) in all patients, including those with ADHD. Standardized DNA extraction and sequencing methods were used to compare gut microbial alpha-diversity among the groups. Dietary diversity was calculated from a standardized dietary questionnaire form. We compared the difference in gut microbiome between patients with ASD and/or ADHD with neurotypical siblings and non-related neurotypical controls. RESULTS: Ninety-eight subjects were included in the study (18 with ASD, 19 with ADHD, 20 with both ASD and ADHD, 13 neurotypical siblings, and 28 non-related neurotypical controls). The alpha-diversity indices, such as Chao 1 and Shannon index, showed a significant difference between the groups in a Linear mixed-effect model (F(4, 93) = 4.539, p = .02), (F(4, 93) = 3.185, p = .017), respectively. In a post-hoc pairwise comparison, patients with ASD had lower alpha-diversity compared with non-related controls after Bonferroni correction. Dietary diversity shown in Shannon index did not differ among the groups (F(4, 84) = 1.494, p = .211). CONCLUSIONS: Our study indicates disorder-specific microbiome differences in patients with ASD. In future research on gut microbiota in neurodevelopmental disorders, it is necessary to consider the impact of ASD and ADHD co-occurrence, and strictly control for background information such as diet, to elucidate the gut-microbiota interaction in ASD and ADHD for exploring the potential of therapeutic interventions.

Enhancement of cerebroprotective effects of lipid nanoparticles encapsulating FK506 on cerebral ischemia/reperfusion injury by particle size regulation.

Delivery of cerebroprotective agents using liposomes has been demonstrated to be useful for treating cerebral ischemia/reperfusion (I/R) injury. We previously reported that intravenous administration of liposomes with diameters of 100 nm showed higher accumulation in the I/R region compared with larger liposomes (>200 nm) by passage through the disintegrated blood-brain barrier, suggesting a size-dependence for liposome-mediated drug delivery. Based on these findings, we hypothesized that regulation of liposomal particle size (<100 nm) may enhance the therapeutic efficacy of encapsulated drugs on cerebral I/R injury. Herein, we prepared lipid nanoparticles (LNP) with particle sizes <100 nm by the microfluidics method and compared their therapeutic potential with LNP exhibiting sizes >100 nm in cerebral I/R model rats. Intravenously administered smaller LNP (ca. 60 nm) exhibited wider accumulation and diffusivity in the brain parenchyma of the I/R region compared with larger LNP (>100 nm). Importantly, treatment with LNP encapsulating the cerebroprotective agent FK506 (FK-LNP) with particle sizes <100 nm showed greater cerebroprotective effects than FK-LNP with sizes >100 nm, and also significantly ameliorated brain injury. These results suggest that particle size regulation of LNP to sizes <100 nm can enhance the therapeutic effect of encapsulated drugs for treatment of cerebral I/R injury, and that FK-LNP could be a promising cerebroprotective agent.

Effective Anticancer Therapy by Combination of Nanoparticles Encapsulating Chemotherapeutic Agents and Weak Electric Current.

Delivery of medicines using nanoparticles via the enhanced permeability and retention (EPR) effect is a common strategy for anticancer chemotherapy. However, the extensive heterogeneity of tumors affects the applicability of the EPR effect, which needs to overcome for effective anticancer therapy. Previously, we succeeded in the noninvasive transdermal delivery of nanoparticles by weak electric current (WEC) and confirmed that WEC regulates the intercellular junctions in the skin by activating cell signaling pathways (J. Biol. Chem., 289, 2014, Hama et al.). In this study, we applied WEC to tumors and investigated the EPR effect with polyethylene glycol (PEG)-modified doxorubicin (DOX) encapsulated nanoparticles (DOX-NP) administered via intravenous injection into melanoma-bearing mice. The application of WEC resulted in a 2.3-fold higher intratumor accumulation of nanoparticles. WEC decreased the amount of connexin 43 in tumors while increasing its phosphorylation; therefore, the enhancing of intratumor delivery of DOX-NP is likely due to the opening of gap junctions. Furthermore, WEC combined with DOX-NP induced a significant suppression of tumor growth, which was stronger than with DOX-NP alone. In addition, WEC alone showed tumor growth inhibition, although it was not significant compared with non-treated group. These results are the first to demonstrate that effective anticancer therapy by combination of nanoparticles encapsulating chemotherapeutic agents and WEC.

A simple, fast, and orientation-controllable technology for preparing antibody-modified liposomes.

Modification with antibodies is a useful strategy for the delivery of nanoparticles to target cells. However, the complexity of the required chemical modifications makes them time-consuming and low efficiency, and the orientation of the antibody is challenging to control. To develop a simple, fast, effective, and orientation-controllable technology, we employed staphylococcal protein A, which can bind to the Fc region of antibodies, as a tool for conjugating antibodies to nanoparticles. Specifically, we modified the C-domain dimer of protein A to contain a lysine cluster to create a molecule, DPACK, that would electrostatically bind to anionic liposomes. Using this protein, antibody-modified liposomes can be prepared in 35 min with two steps: (1) interaction of DPACK with liposomes and (2) interaction of an antibody with DPACK-modified liposomes. Binding efficiencies of DPACK with liposomes and IgG with DPACK-modified liposomes were 75% and 72-84%, respectively. Uptake of liposomes modified with anti-epidermal growth factor receptor (EGFR) antibodies via DPACK by EGFR-expressing cancer cells was significantly higher than that of unmodified liposomes, and the liposomes accumulated in tumors and colocalized with EGFR. This simple, fast, effective and orientation-controllable technology for preparing antibody-modified liposomes will be useful for active targeting drug delivery.

Gastrointestinal symptoms and sensory abnormalities associated with behavioral problems in children with neurodevelopmental disorders.

Behavioral problems directly affect the quality of life of caregivers and children with autism spectrum disorder (ASD) and/or attention-deficit/hyperactivity disorder (ADHD), and is known to be associated with clinical factors such as gastrointestinal (GI) symptoms, sensory abnormalities, intellectual abilities, and use of medication. However, previous studies have not considered these relationships comprehensively. We conducted a cross-sectional study of 6-12-year-old children with diagnoses of ASD and/or ADHD at two hospitals in Japan. Scores for the aberrant behavior checklist (ABC), autism-spectrum quotient (AQ), and Conners 3, as well as information on daily sleep and exercise, GI symptoms, and Short Sensory Profile, were collected. Each factor was subjected to a correlation analysis to investigate its effect on ABC scores. A stepwise multiple linear regression analysis for the factors with p < 0.05 was performed. Data were obtained from 60 patients with a mean age of 8.3 years; 21 had ASD alone, 18 had ADHD alone, and 21 had ASD + ADHD. The correlation analyses identified six factors associated with ABC severity: (a) methylphenidate use, (b) Conners hyperactivity score, (c) Conners inattention score, (d) AQ score, (e) SSP score, and (f) GI symptom score. The multiple regression showed that "GI symptoms" and "sensory abnormalities" were independently associated with ABC severity. Although further studies are needed to show a causal relationship, appropriate assessment of GI symptoms and sensory abnormalities may help alleviate some problematic behaviors and improve the quality of life of children with neurodevelopmental disorders and their families. LAY SUMMARY: Behavioral problems in children with neurodevelopmental disorders are known to be associated with many factors. This study aimed to comprehensively investigate the known factors. We have discovered that "gastrointestinal symptoms" and "sensory abnormalities" were independently associated with Behavioral problems. Our results suggest that it is important for clinicians and caregivers to pay more attention to children's GI symptoms and sensory abnormalities that may not present as obvious symptoms or complaints.

Weak Electric Current Treatment to Artificially Enhance Vascular Permeability in Embryonated Chicken Eggs.

Technologies that overcome the barrier presented by vascular endothelial cells are needed to facilitate targeted delivery of drugs into tissue parenchyma by intravenous administration. We previously reported that weak electric current treatment (ET: 0.3-0.5 mA/cm2) applied onto skin tissue in a transdermal drug delivery technique termed iontophoresis induces cleavage of intercellular junctions that results in permeation of macromolecules such as small interfering RNA and cytosine-phosphate-guanine (CpG) oligonucleotide through the intercellular space. Based on these findings, we hypothesized that application of ET to blood vessels could promote cleavage of intercellular junctions that artificially induces increase in vascular permeability to enhance extravasation of drugs from the vessels into target tissue parenchyma. Here we investigated the effect of ET (0.34 mA/cm2) on vascular permeability using embryonated chicken eggs, which have blood vessels in the chorioallantoic membrane (CAM), as an animal model. ET onto the CAM of the eggs significantly increased extravasation of intravenously injected calcein (M.W. 622.6), a low molecular weight compound model, and the macromolecule fluorescein isothiocyanate (FITC)-dextran (M.W. 10000). ET-mediated promotion of penetration of FITC-dextran through vascular endothelial cells was also observed in transwell permeability assay using monolayer of human umbilical vein endothelial cells without induction of obvious cellular damage. Confocal microscopy detected remarkable fluorescence derived from injected FITC-dextran in blood vessel walls. These results in embryonated chicken eggs suggest that ET onto blood vessels could artificially enhance vascular permeability to facilitate extravasation of macromolecules from blood vessels.