Single-cell RNA-seq reveals altered NK cell subsets and reduced levels of cytotoxic molecules in patients with ankylosing spondylitis.

Ankylosing spondylitis (AS) is an autoimmune disease with unknown aetiology. To unravel the mechanisms mediating AS pathogenesis, we profiled peripheral blood mononuclear cells (PBMCs) from AS patients and healthy subjects using 10X single-cell RNA sequencing. The frequencies of immune cell subsets were evaluated by flow cytometry. NK cells were purified from PBMCs using isolation kit and were examined for gene expression by RT-qPCR. Plasma levels of cytolytic molecules were examined by enzyme-linked immunosorbent assay. Compared to healthy controls, AS patients showed a significant decrease in total NK cells as well as CD56dim NK subset, whereas CD56bright NK cells were increased. Additionally, impaired expression of cytotoxic genes in NK cells of AS patients was observed by bioinformatics algorithm and verified by RT-qPCR and flow cytometry. Consistent with changes in transcriptomics, we found decreased plasma levels of granzymes, but not granulysin, in AS patients. Furthermore, Pearson correlation analysis revealed a negative correlation between plasma GZMB levels and disease activity (r = -0.5275, p = 0.0358). No correlation was observed between plasma cytolytic molecules and biochemical indexes (ESR and CRP). Our findings uncover altered NK cell subsets and cytotoxic profiles in peripheral circulation of AS patients at single-cell resolution.

Another new attempt to repair finger pulp defects: retrograde island flap bridge transfer of the adjacent phalangeal artery combined with vascular pedicle tubular skin grafting.

This study investigated the surgical method and therapeutic effect of retrograde island flap bridge transfer of the adjacent phalangeal artery combined with vascular pedicle tubular skin grafting to repair finger pulp defects. From June 2008 to May 2020, 21 fingers (19 patients) were repaired using this method. The postoperative flap survival rate and complications, and the clinical effect, were evaluated. All flaps survived, and all patients were followed-up for 12 to 46 months. The static two-point discrimination (2PD) was 7 to 11 mm, no apparent complications were observed in the donor area and the McIndoe cold intolerance symptom severity (CISS) scores indicated mild severity. The Michigan hand outcome questionnaire (MHQ) indicated that all patients were satisfied with their overall hand appearance and function. Results were excellent in 15 cases and good in 4 cases, according to the Dargan function evaluation (DFE). It is safe and effective to repair finger pulp defects with a retrograde island flap bridge transfer of the adjacent phalangeal artery combined with vascular pedicle tubular skin grafting. This skin flap has the advantages of simple severing, good texture and concealed donor area, which is convenient for early postoperative functional exercise of the finger.

CD96 Downregulation Promotes the Immune Response of CD4 T Cells and Associates with Ankylosing Spondylitis.

Inhibitory receptors (IRs) play an indispensable role in regulating T cell activation and expansion. This study is aimed at exploring the correlation between IRs and ankylosing spondylitis (AS). Bioinformatics analysis of two datasets (GSE25101 and GSE73754), including 68 AS cases and 36 healthy controls, demonstrated that "T cell receptor signaling pathway" was significantly enriched, and two IRs (CD112R and CD96) were downregulated in AS cases. Real-time Quantitative PCR Detecting System (qPCR) analysis confirmed the decreased expression of CD112R and CD96 in the peripheral blood of AS patients. Flow cytometry demonstrated that the frequency of CD96-positive cells among CD4 T cells in AS patients was significantly reduced and that expressed on the cells was also significantly lower than the healthy controls. In addition, the expression of CD96 was altered on human primary CD4 T cells extracted from 3 healthy volunteers and cocultured with allogeneic dendritic cells (DCs). Also, low expression of CD96 elevated the phosphorylation of ERK in CD4 T cells and increased the level of TNF-α, IL-23, IL-17A, IL-6, and IFN-γ in the cell culture supernatant. These results suggested that CD96 is crucial for the pathogenesis of AS and may be a potential target in the treatment of the disease.

Efficacy and Safety of Wenbu Zhibi Granule in Patients with Ankylosing Spondylitis: A Multicenter, Randomized, Double-blind, Placebo-controlled Trial.

Background. Ankylosing spondylitis (AS) is a chronic disease in which the column is the main lesion. It is caused by a combination of genetic and environmental factors, mainly involving the axial skeleton, resulting in column rigidity and difficulty in movement, and there may be different degrees of eye, lung, cardiovascular, kidney, and other organ damage. Long-term treatment lacks in ankylosing spondylitis. Wenbu Zhibi granule (WZG) is a prescription handed down from the history of Chinese medicine for thousands of years, which is used to treat the pain of patients with AS and to prevent the further development of the disease. However, there is no scientific evidence based on clinical trials to evaluate the efficacy and safety of WZG for ankylosing spondylitis. Methods/Design. We will conduct a multicenter, randomized, double-blind, placebo-controlled trial to evaluate the efficacy and safety of the WZG in the treatment of AS. We will randomly assign 100 patients with active AS to two groups, treated for 16 weeks. The primary efficacy endpoint is the proportion of subjects who reached 40% improvement criteria proposed by Assessment of SpondyloArthritis International Society (ASAS40) at 16 weeks from baseline, the secondary efficacy endpoint includes ASAS20 response rate, ASAS partial remission response rate, 5/6 improvement criteria proposed by ASAS (ASAS5/6) response rate, and change in the Spondyloarthritis Research Consortium of Canada (SPARCC) MRI spine score, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Ankylosing Spondylitis Disease Activity Score (ASDAS), linear Bath Ankylosing Spondylitis Metrology Index (BASMI), ankylosing spondylitis quality of life (ASQoL). In addition, the time points will be set as baseline, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 24 weeks, and 48 weeks. Discussion. The results of this study will elucidate the efficacy and safety of WZG and provide an appropriate treatment option for patients with AS. Trial registration: ClinicalTrials.gov ID: https://clinicaltrials.gov/ct2/show/ChiCTR2000041010. (Chinese Clinical Trail Registry, Registered 16 December 2020, http://www.chictr.org.cn).

Identification of immune related cells and crucial genes in the peripheral blood of ankylosing spondylitis by integrated bioinformatics analysis.

BACKGROUND: Ankylosing spondylitis (AS) is a progressive rheumatic disease and studies reveal that the immune system is critical for the pathogenesis of AS. In the present study, various bioinformatics analysis methods were comprehensively applied, designed to identify potential key genes and inflammation states of AS. METHODS: The transcriptome profiles of GSE25101 and GSE73754 obtained from the Gene Expression Omnibus (GEO) database were merged for subsequent analyses. The differentially expressed genes (DEGs) were identified using the Bioconductor package Limma and threshold values. Functional enrichment and pathway enrichment analyses were performed using the clusterProfiler package and Gene Set Enrichment Analysis (GSEA). Next, protein-protein interaction (PPI) network of the identified DEGs was constructed by the online database, the Search Tool for the Retrieval of Interacting Genes (STRING), visualization and analysis were performed through Cytoscape software. Subsequently, we applied CIBERSORT algorithm to identify subpopulation proportions of immune cells in peripheral blood samples. Finally, we validated the hub genes with the GSE18781 dataset. Samples were collected from patients to validate gene and protein expression using qRT-PCR and ELISA. RESULTS: A total of 334 DEGs were identified, including 182 upregulated and 152 downregulated DEGs, between AS patients and normal human controls, which were primarily involved in immune response, autophagy, and natural killer cell-mediated cytotoxicity. The most prominent module and candidate biomarkers were identified from the PPI network. Biomarkers were selected for validation and their expressions were significantly decreased in peripheral blood samples which was consistent with transcriptome sequencing results. Nine genes with AUC > 0.70 were considered to be AS hub genes for ROC curve analysis, including GZMA, GZMK, PRF1, GNLY, NKG7, KLRB1, KLRD1, IL2RB and CD247. Furthermore, CIBERSORT results suggest that AS contained a higher proportion of CD8+ T cells, naive CD4+ T cells, neutrophils, and lower levels of gamma delta T cells compared with the normal controls. CONCLUSION: In this study, we identified DEGs combined with their closely related biological functions and propose that granule-associated proteins and immune infiltration maybe involved in the progression of ankylosing spondylitis. These validated hub genes may provide new perspectives for understanding the molecular mechanisms of ankylosing spondylitis.

[Whole exome sequencing in a pedigree with ankylosing spondylitis].

OBJECTIVE: To choose the disease-causing gene in a Chinese pedigree with ankylosing spondylitis (AS) by whole-exome sequencing (WES), and provide theory basis for mechanism of disease. METHODS: Clinical data of AS pedigree were collected, including 2 males, the age were 48 and 18 years old, the course of disease were 23 and 4 years. Whole blood genomic DNA of AS was extracted to perform whole exome sequencing, the results were compared with human databases, common variations which had been reported were wiped out, then non synonymous single nucleotide variants(SNVs) from the family members were combined, and candidate genes was selected initially. RESULTS: Totally 80 G data was obtained from AS family with high quality.By comparing results between patient and normal subject, and filtering with number of biological database, the result showed heterozygous mutation of JAK2 gene 12 exon c.1709 A>G (p.Tyr570Cys) may be the potential disease-causing gene. The variant c.1151T>C of MUC3A gene may be one of the causes of intestinal symptoms in the family members. CONCLUSION: It is feasible to find t candidate gene mutations of AS by Exon sequencing. The mutation c.1709 A>G in gene JAK2 identified by whole exome sequencing might be the pathogenic mutation in this AS pedigree.