Gastrointestinal symptoms are more prevalent in children with autism spectrum disorder (ASD) than in typically developing (TD) children. Constipation is a significant gastrointestinal comorbidity of ASD, but the associations among constipated autism spectrum disorder (C-ASD), microbiota and short-chain fatty acids (SCFAs) are still debated. We enrolled 80 children, divided into the C-ASD group (n = 40) and the TD group (n = 40). In this study, an integrated 16S rRNA gene sequencing and gas chromatography–mass spectrometry-based metabolomics approach was applied to explore the association of the gut microbiota and SCFAs in C-ASD children in China. The community diversity estimated by the Observe, Chao1, and ACE indices was significantly lower in the C-ASD group than in the TD group. We observed that Ruminococcaceae_UCG_002, Erysipelotrichaceae_UCG_003, Phascolarctobacterium, Megamonas, Ruminiclostridium_5, Parabacteroides, Prevotella_2, Fusobacterium, and Prevotella_9 were enriched in the C-ASD group, and Anaerostipes, Lactobacillus, Ruminococcus_gnavus_group, Lachnospiraceae_NK4A136_group, Ralstonia, Eubacterium_eligens_group, and Ruminococcus_1 were enriched in the TD group. The propionate levels, which were higher in the C-ASD group, were negatively correlated with the abundance of Lactobacillus taxa, but were positively correlated with the severity of ASD symptoms. The random forest model, based on the 16 representative discriminant genera, achieved a high accuracy (AUC = 0.924). In conclusion, we found that C-ASD is related to altered gut microbiota and SCFAs, especially decreased abundance of Lactobacillus and excessive propionate in faeces, which provide new clues to understand C-ASD and biomarkers for the diagnosis and potential strategies for treatment of the disorder. This study was registered in the Chinese Clinical Trial Registry (www.chictr.org.cn; trial registration number ChiCTR2100052106; date of registration: October 17, 2021).

Helicobacter pylori (H. pylori) is a microaerophilic, gram-negative spirochete that primarily colonizes the human gastric mucosa. It is strongly linked to gastritis, ulcers, and the development of malignant tumors. Macrophages, as one of the key components of the innate immune system, play a crucial role in maintaining immune homeostasis through a range of functions, including pathogen phagocytosis, antigen recognition and presentation, inflammation regulation and tumor immune surveillance. Emerging evidence suggests that H. pylori employs diverse molecular mechanisms to evade immune clearance by macrophages. This review provides a comprehensive analysis of how H. pylori infection modulates macrophage functions, including impairing pathogen recognition and phagocytosis, disrupting phagosome maturation and reducing immune clearance capacity. Furthermore, H. pylori infection skews macrophage polarization to promote chronic inflammatory damage, inhibits antigen processing and presentation to evade adaptive immune responses and induces macrophage apoptosis via activation of apoptotic signaling pathways. By unraveling the complex molecular interactions between H. pylori and macrophages, this review highlights strategies for reprogramming macrophage functions, offering innovative approaches to address the limitations of conventional antimicrobial therapies and advancing targeted therapeutic interventions for H. pylori-associated diseases.

Background: The preparation method of fecal microbiota suspensions is crucial for reliable fecal microbiota transplantation research. However, current protocols vary significantly in processing parameters, potentially compromising the comparability of studies. Systematic comparisons of how different preparation techniques affect the microbial community are still lacking. Methods: Fresh fecal samples from nine adult volunteers were processed via filtration and centrifugation at various speeds. Microbial viability was assessed via live/dead staining and colony forming unit enumeration. 16S rDNA sequencing was performed to analyze bacterial diversity and taxonomic composition. Results: The microbial composition and functional potential did not differ between the filtration alone group and the fresh fecal group, and filtration resulted in the lowest bacterial mortality. Mortality increased with increasing centrifugation speed. Centrifugation selectively affected the abundance of some genera (e.g., Delftia and Acinetobacter ). High-speed supernatants presented markedly lower OD ₆₀₀ values than low-speed supernatants did, and differential centrifugation visibly reduced the amount of debris. Conclusion: Filtration alone best preserved fecal microbial viability, composition and functional potential. Centrifugation enrichment becomes selective at high speeds. Differential centrifugation offered superior impurity removal. The preparation strategy should be tailored to the research or therapeutic goal.

Background: Migraine, a prevalent and debilitating neurovascular disorder, frequently co-occurs with functional dyspepsia. Emerging evidence suggests that Helicobacter pylori (H. pylori) infection may contribute to both conditions via neuroimmune pathways, though the molecular basis for this association has yet to be fully elucidated. Methods: H. pylori -related datasets from GEO were analyzed to identify differentially expressed genes (DEGs), followed by functional enrichment analyses including GO, KEGG, GSEA, and GSVA. Immune infiltration patterns were assessed, and CGRP-related hub genes were identified using machine learning approaches. The relationship between these hub genes and immune cell infiltration—particularly M1 macrophage polarization—was explored, and the interaction between CALCA and PNOC was analyzed by correlation analysis and protein-protein interaction networks. Experimental validation was performed using RT-qPCR in H. pylori -infected THP-1 macrophages and SH-SY5Y neuronal cells, along with loss- and gain-of-function experiments to investigate regulatory relationships. Results: Transcriptomic analysis of H. pylori -infected patients identified 683 differentially expressed genes enriched in immune and inflammatory pathways. Immune profiling further revealed prominent M1 macrophage polarization with increased γδT cells and B lymphocytes. Among the DEGs, four CGRP-related hub genes (PNOC, ICAM1, MMP9, and NFE2L1) were identified and found upregulated in H. pylori -infected macrophages. Correlation analyses showed PNOC, ICAM1, and MMP9 positively associated with M1 macrophages and their canonical markers, with knockdown of each gene attenuating M1 marker expression. In contrast, NFE2L1 showed no significant correlation, yet its knockdown increased M1 marker expression. Among the hub genes, only PNOC correlated positively with CALCA; both were upregulated in H. pylori -infected neuronal cells. Notably, PNOC knockdown in macrophages reduced CALCA expression in co-cultured neurons, while overexpression had the opposite effect, suggesting macrophage-derived PNOC may regulate neuronal CGRP expression. Conclusion: H. pylori infection may promote migraine and functional dyspepsia through gastric inflammation and neuroimmune signaling. This process involves four key genes—PNOC, ICAM1, MMP9, and NFE2L1—that regulate M1 macrophage polarization and pro-inflammatory responses. Among them, PNOC appears to link gastric inflammation to neurological symptoms by modulating neuronal CGRP. Together, these findings point to gut-brain axis involvement in this comorbidity and may inform future therapies targeting H. pylori eradication and CGRP-related pathways.