Effects of small molecule inhibitor SW033291 on hepatic ischemia-reperfusion injury in mice.

Hepatic ischemia-reperfusion injury (IRI) is an important factor in the recovery of liver function and prognosis after liver surgery. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) small molecule inhibitor SW033291 is thought to play an important role in promoting tissue regeneration. The aim of this study was to investigate the effect of 15-PGDH on ischemia-reperfusion injury in mouse liver. In this study, mice in the administered group showed lower necrosis and inflammation than control mice, partly due to reduced macrophage and neutrophil infiltration in liver tissue, as well as reduced serum or tissue levels of pro-inflammatory cytokines and oxidative stress. In addition, mice in the administered group showed increased post-injury reparation due to IRI-induced injury compared to control mice. CONCLUSION: The small molecule inhibitor SW033291 played a better role in preventing hepatic ischemia-reperfusion injury in mice and may be a potential option to prevent the development and progression of hepatic I/R.

Identification of Cofilin-1 as a novel biomarker of atopic dermatitis using iTRAQ quantitative proteomics.

BACKGROUND: Atopic dermatitis (AD) is a chronic relapsing inflammatory skin condition; however, little is known about the pathogenesis and serum biomarker of this disease. METHODS: Isobaric tagging for relative and absolute quantitation (iTRAQ) proteomic assay was adopted to identify and quantify the differentially expressed proteins (DEPs) in the serum of AD patients. Bioinformatic analysis, including GO, Reactome, GSEA, PPI, and ssGSEA analysis, were used to identified the enriched pathways, hub proteins and immune cells. The expression level and distribution of hub proteins were confirmed by ELISA and IHC. RESULTS: Sixty-six DEPs were identified with iTRAQ proteomic assay by analyzing serum from AD patients and normal subjects. GO and Reactome analysis shown the alternated pathway were mainly involved in immunity, oxidative stress, and actin cytoskeleton. The GSEA and PPI network analysis among the DEPs were carried out and identified Cofilin-1 and profilin-1 as the core components of this network. Additionally, the disruption of Th1/Th2/Th17 cell balance and the significantly reducing of Treg, MDSC, and γδT cells was also found in AD patients using the ssGSEA analysis. Further ELISA and IHC assay validated the significantly elevated expression of Cofilin-1 in AD patients. CONCLUSION: Our results suggested that Cofilin-1 may serve as a novel biomarker for AD diagnosis.

Design and functional preliminary investigation of recombinant antigen EgG1Y162-EgG1Y162 against Echinococcus granulosus.

In the early stage, our research group cloned Echinococcus granulosus-specific antigen, EgG1Y162, from protoscolex and adult worms of E. granulosus. In order to enhance the immunogenicity of the vaccine, we prepared a recombinant vaccine by tandemly linking EgG1Y162, splicing the protein and linker at the gene level. This approach is expected to improve the immunogenicity of the vaccine by enhancing the molecular weight of the protein and increasing the antigenic epitopes. Bioinformatics was used to predict the physicochemical properties, transmembrane domain, protein structure, and T-/B-cell antigenic epitope of different recombinant proteins, EgG1Y162-linker-EgG1Y162. Finally, the linker sequence, "GGGGSGGG," which had the least influence on the migration of recombinant protein T/B epitope and can fold normally in series with EgG1Y162, was selected to design the recombinant vaccine. The plasmid was produced using genetic engineering techniques, and the recombinant protein, EGG1Y162-GGGGSGGG-EgG1Y162, was induced to be expressed and purified. EgG1Y162-GGGGSGGG-EgG1Y162 was identified to be correctly expressed with 100% specificity. Compared with EgG1Y162, EgG1Y162-GGGGSGGG-EgG1Y162 was more likely to promote dendritic cell maturation. EgG1Y162-GGGGSGGG-EgG1Y162 was speculated to have the potential to improve antigen immunogenicity by increasing the molecular weight and antigenic epitope.

Cloning and identification of the CTLA-4IgV gene and functional application of vaccine in Xinjiang sheep.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an important surface molecule of activated T cells that has a strong affinity with the B7 molecule on the surface of antigen-presenting cells. Among these molecules, the CTLA-4 extracellular region (CTLA-4 IgV) may be used as a novel immune adjuvant molecule for delivering antigens and inducing strong humoral and cellular immune responses. In this study, bioinformatics analysis was performed to determine and clone the extracellular region of Xinjiang sheep CTLA-4 (NM_001009214). The CTLA-4 IgV gene was amplified and ligated into the pMD19-T vector, and the positive bacteria were screened by blue-white spots for sequencing and comparison. The correctly sequenced CTLA-4 IgV was digested and then ligated into the prokaryotic expression vector pET-30a(+). The plasmid pET30a-CTLA-4 IgV was constructed to induce the expression of the recombinant protein CTLA-4 IgV. Thereafter, CTLA-4 IgV was identified. Clustal X multiple sequence alignment revealed that the protein sequence of Xinjiang sheep CTLA-4 IgV was different from that of the known CTLA-4 extracellular region. The 3D protein structure of Xinjiang sheep CTLA-4 IgV was constructed via the bioinformatics method. Subsequently, molecular docking between the Xinjiang sheep CTLA-4 IgV protein and the B7 molecule was conducted. Results revealed multiple binding sites in the extracellular region of Xinjiang sheep CTLA-4, and two multiple interactions ensured stable binding after docking. The functionality of the Xinjiang sheep CTLA-4 IgV protein was further verified by fusing the CTLA-4 extracellular V region with EgG1Y162, a protective protein from Echinococcus granulosa, and the purified recombinant protein CTLA-4 IgV-EgG1Y162 was expressed with the mouse bone marrow-derived. The addition of the Xinjiang sheep CTLA-4 IgV protein at the amino terminus promoted the binding of EgG1Y162 to dendritic cells (DCs) and increased the maturation rate of these cells, further indicating that the protein could effectively improve the antigen presentation ability of DCs. The CTLA-4 extracellular domain protein of Xinjiang sheep is unique and has the potential to promote the presentation of the fusion protein by DCs as an adjuvant. The cloning and expression of this gene provide new measures and ideas for the preparation of the Xinjiang sheep vaccine to prevent zoonotic diseases.

PD-1 Blockade Restores the Proliferation of Peripheral Blood Lymphocyte and Inhibits Lymphocyte Apoptosis in a BALB/c Mouse Model of CP BVDV Acute Infection.

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocytes (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the situation in vivo remains to be further studied and confirmed. Therefore, in this study, we established a BALB/c mouse model of acute BVDV infection with cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain NY-1). Then, we examined the mRNA and protein levels of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC) from BVDV-infected mice and analyzed the effects of PD-1 blockade on the proportions of CD3+, CD4+, and CD8+ T cell subsets, the apoptosis and proliferation of PBL, and the production of IL-2 and IFN-γ. We found that leukopenia, lymphocytopenia, and thrombocytopenia were developed in both CP and NCP BVDV-infected mice at day 7 of post-infection. The mRNA and protein expression of PD-1 and PD-L1 were significantly upregulated in CP and NCP BVDV-infected mice. Moreover, PD-1/PD-L1 upregulation was accompanied by leukopenia and lymphopenia. Additionally, PD-1 blockade inhibited PBL apoptosis and virus replication, restored the proportions of CD3+, CD4+, and CD8+ T cell subsets, and increased IFN-γ production and p-ERK expression in BVDV-infected mice. However, blocking PD-1 did not significantly affect PBL proliferation and IL-2 production in NCP BVDV-infected mice. Our findings further confirmed the immunomodulatory role of PD-1 in peripheral blood lymphocytopenia in vivo and provided a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

PD-1 blockade inhibits lymphocyte apoptosis and restores proliferation and anti-viral immune functions of lymphocyte after CP and NCP BVDV infection in vitro.

Bovine viral diarrhea virus (BVDV) is an important virus that can cause extensive economic losses in both dairy and beef industry worldwide. Acute infection with BVDV results in peripheral blood lymphopenia, apoptosis and immunosuppression. Up-regulated programmed death-1 (PD-1) expression induces functional exhaustion of lymphocytes, inhibition of proliferation and apoptosis of lymphocytes during acute and chronic viral infections, such as HIV and HCV. However, there are no reports showing the role of PD-1 in peripheral blood lymphopenia, apoptosis and immunosuppression after acute BVDV infection. Accordingly, we measured the mRNA and protein expression of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) infected with BVDV, and analyzed the effects of PD-1 blockade on immune-associated function and activity in peripheral blood lymphocytes (PBLs). The results showed that both cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection stimulated the mRNA and protein expression of PD-1 and PD-L1 significantly. The upregulation of PD-1/PD-L1 was accompanied by the decreased PBLs proliferation and increased apoptosis. Additionally, PD-1 blockade restored proliferation, inhibited apoptosis, increased IFN-γ production and decreased BVDV load. Remarkably, the PD-1/PD-L1 interaction has a more substantial effect on the immunoregulation of inhibiting proliferation induced by CP BVDV infection. Our findings confirm that PD-1 plays a vital role in peripheral blood lymphopenia and apoptosis caused by acute BVDV infection, and provide new insights into exploring the immunopathological mechanisms of BVDV or other members of the Flaviviridae family, and a potential therapeutic strategy to control BVDV infection.