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Background: Primary biliary cholangitis (PBC) is a chronic intrahepatic cholestatic autoimmune liver disease characterized by inflammatory injury of small and medium-sized bile ducts in the liver. The pathogenesis of PBC has yet to be entirely understood. CD47/signal-regulatory protein alpha (SIRPα) is closely related to developing autoimmune diseases by promoting inflammatory response. However, the effect of CD47/SIRPα on inflammatory response in PBC patients is still unclear., Objective: We investigated the expression of CD47/SIRPα and the effect of inflammatory cytokines on the CD47 expression, analyzed potential autoantibodies against CD47 and the effect of anti-CD47 antibody on the inflammatory response in PBC, provided laboratory basis for the study of the pathogenesis and targets for non-invasive diagnosis and treatment on PBC., Methods: The expression levels of CD47 and SIRPα on peripheral blood mononuclear cells (PBMC) were measured in 14 patients with PBC (the PBC group) and 13 healthy subjects (the Control group) by flow cytometry (FCM). The PBMC derived from healthy subjects were stimulated with healthy subjects' serum, PBC patients' serum, IFN-α or TNF-α, and the CD47 expression level on CD14 + monocytes was detected by FCM. The level of serum anti-CD47 antibody or IFN-α in PBC patients and healthy subjects was analyzed by ELISA. FCM was used to examine the TNF-α expression level in CD14 + monocytes of healthy subjects stimulated with isotype control antibody, anti-CD47 antibody, LPS or LPS combined with CD47 antibody., Results: The CD47 expression level on the CD14 + monocytes in PBC patients was statistically higher than that in the Control group ( P <0.01). Compared with the Control group (PBMC+healthy serum), the CD47 expression on CD14 + monocyte stimulated with the PBC patients' serum (PBMC+PBC patients' serum) was increased ( P <0.001); the CD47 expression on CD14 + monocyte stimulated with IFN-α (PBMC + IFN-α) increased gradually with the increased concentration of IFN-α ( P <0.05). However, there was no similar trend on CD14 + monocyte stimulated with the TNF-α (PBMC+TNF-α) ( P >0.05). The levels of serum anti-CD47 antibody and IFN-α in the PBC patients were higher than those in healthy subjects ( P <0.05). The TNF-α expression level in CD14 + monocyte stimulated with the LPS (PBMC+LPS) or anti-CD47 antibody+LPS group (PBMC+LPS+anti-CD47 antibody) was significantly increased than that in the Control group (PBMC+isotype control antibody) ( P <0.01 and P <0.001, respectively). The TNF-α expression level in CD14 + monocyte stimulated with the anti-CD47 antibody + LPS was higher than that with the LPS ( P < 0.05)., Conclusion: The CD47 may be related to the pathogenesis of PBC by inflammatory response. The CD47/SIRPα signal were imbalanced in PBC patients. The presence of serum anti-CD47 antibodies in PBC patients provides a laboratory basis for clinical diagnosis and treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Su, Jin, Liu, Zhu and Li.)

We constructed a recombinant plasmid containing the N-terminus gene of vacA gene of Helicobacter pylori and studied the effect of VacA on the secretion of macrophages as an individual virulence determinant. VacA gene amplified by Polymerase Chain Reaction (PCR) from Helicobacter pylori was cloned into eukaryotic expression vector pDsRed-Monomer-C1. The recombinant plasmids were verified by restriction endonucleases analysis and nucleotide sequencing. Then the recombinant plasmids pDsRed-Monomer-C1/vacA were transfected into macrophages. Their expression in macrophages was examined by Western blot and fluorescence microscope. Vacuolated phenotype in macrophages was observed by electron microscopy and neutral red uptake. The cytokine content of TNF-alpha or IL-1beta in the culture medium was tested quantitatively with Enzyme Linked Immunosorbent Assay (ELISA) kit, respectively. The effect of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, on the secretion of macrophages transfected with the recombinant plasmids, was also studied. Restriction endonucleases analysis and nucleotide sequencing showed that the eukaryotic expression recombinant pDsRed-Monomer-C1/vacA was successfully constructed. A clear vacuolated phenotype developed in some of macrophages transfected with the recombinant plasmids. VacA over-expressed increased the level of TNF-alpha and IL-1beta. PDTC decreased the production of TNF-alpha and IL-1beta induced by VacA. In conclusion, we have successfully constructed the eukaryotic expression plasmid encoding VacA. The over-expression of VacA fusion protein can up-regulate secretion of macrophages. Activation of NF-kappaB is probably involved in VacA induced cytokines production.