Inflammation and the gut-brain axis have been implicated in the pathogenesis of autism spectrum disorders (ASDs). To further understand the relationship between aberrant immune responses and dysbiotic features of the gut microbiome in ASD, we enrolled 45 ASD individuals and 41 healthy control subjects with ages ranging from 2 to 19 years. We found that ASD group subjects have significantly higher plasma levels of IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, TNF-β, and IFN-γ when compared to healthy controls (FDR-adjusted p < 0.05). The plasma levels of pro-inflammatory cytokines IFN-γ and IL-6 are found to be further associated with several largely pathogenic gut microbiota uniquely detected in subjects with ASD. Furthermore, the ASD gut microbiome is characterized by reduced levels of several beneficial microbiota, including Bacteroides (FDR-adjusted p < 0.01) and Lachnospiraceae (FDR-adjusted p < 0.001). Analysis of Lachnospiraceae family and genus level taxa suggested that relative abundances of such taxa are negatively correlated with pro-inflammatory signaling cytokines IFN-γ and IL-6, particularly in subjects with severe ASD as defined by CARS (p < 0.05). Several largely pathogenic genera are determined to be associated with the pro-inflammatory cytokines IFN-γ and IL-6 (FDR-adjusted p < 0.1). Additionally, IL-4 is significantly negatively correlated with CARS total score (p < 0.05). Based on such results, we propose that the association between the disturbances of specific cytokines and alterations in gut microbiota abundance observed in children and adolescents with ASD provides additional evidence on the induction of aberrant pro-inflammatory mechanisms in ASD and its early diagnosis.

Background: Previous studies have shown that the neurological damage caused by middle cerebral artery occlusion (MCAO) is not only limited to local infarction but can also cause secondary damage in distant sites, such as the hypothalamus. 5-hydroxytryptamine (5-HT)/ 5-HT transporter (5-HTT) and 5-HT receptor 2A (5-HT2A) are important in the treatment of cerebrovascular diseases. Objective: This study aimed to study the effect of electroacupuncture (EA) on the expression of 5-HT, 5-HTT, and 5-HT2A in the hypothalamus of rats with ischemic brain injury and to explore the protective effect and potential mechanism of EA on the secondary injury of cerebral ischemia. Methods: Sprague-Dawley (SD) rats were randomly divided into three groups: sham group, model group, and EA group. The permanent middle cerebral artery occlusion (pMCAO) method was used to induce ischemic stroke in rats. In the EA group, the Baihui (GV20) and Zusanli (ST36) points were selected for treatment, which was administered once per day for two consecutive weeks. The neuroprotective effect of EA was evaluated by nerve defect function scores and Nissl staining. The content of 5-HT in hypothalamus was detected by enzyme linked immunosorbent assay (ELISA), and the expression of 5-HTT and 5-HT2A were detected by Western blot. Results: Compared with that in the sham group, the nerve defect function score in the model group rats was significantly increased, the hypothalamus tissue showed obvious nerve damage, the levels of 5-HT and the expression of 5-HTT were significantly reduced, and the expression of 5-HT2A was significantly increased. After 2 weeks of EA treatment, the nerve defect function scores of pMCAO rats were significantly reduced, the hypothalamic nerve injury was significantly reduced, the levels of 5-HT and the expression of 5-HTT were significantly increased, and the expression of 5-HT2A was significantly decreased. Conclusion: EA has a certain therapeutic effect on hypothalamic injury secondary to permanent cerebral ischemia, and its potential mechanism may be closely related to the upregulation of 5-HT and 5-HTT expression and the downregulation of 5-HT2A expression.

Aims: We aim to define parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS. Background: Engineered, attenuated Herpes simplex virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors have not been fully understood. Objective: This study aimed to construct attenuated HSV-1 vectors using the CRISPR-Cas9 system and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety. Method: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and constructed two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in-vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in the mouse hippocampus gene transduction model. Result: The in-vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacked Poly (A), which induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation. Conclusion: Our results indicated that the integrity of LAT transcripts was not necessary for the establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, suggesting an important role of LAT in maintaining viral reactivation potential.