Autoimmune Diseases ; immunology ; Gene Expression Regulation ; immunology ; Humans ; Immunoglobulin G ; genetics ; immunology ; Sclerosis ; genetics ; immunology ; pathology

Immunoglobulin G4-related sialadenitis (IgG4-RS) is an immune-mediated fibro-inflammatory disease and the pathogenesis is still not fully understood. The aim of this study was to explore the role and mechanism of interleukin-13 (IL-13) in the cellular senescence during the progress of IgG4-RS. We found that the expression of IL-13 and IL-13 receptor α1 (IL-13Rα1) as well as the number of senescent cells were significantly higher in the submandibular glands (SMGs) of IgG4-RS patients. IL-13 directly induced senescence as shown by the elevated activity of senescence-associated β-galactosidase (SA-β-gal), the decreased cell proliferation, and the upregulation of senescence markers (p53 and p16) and senescence-associated secretory phenotype (SASP) factors (IL-1β and IL-6) in SMG-C6 cells. Mechanistically, IL-13 increased the level of phosphorylated signal transducer and activator of transcription 6 (p-STAT6) and mitochondrial-reactive oxygen species (mtROS), while decreased the mitochondrial membrane potential, ATP level, and the expression and activity of superoxide dismutase 2 (SOD2). Notably, the IL-13-induced cellular senescence and mitochondrial dysfunction could be inhibited by pretreatment with either STAT6 inhibitor AS1517499 or mitochondria-targeted ROS scavenger MitoTEMPO. Moreover, IL-13 increased the interaction between p-STAT6 and cAMP-response element binding protein (CREB)-binding protein (CBP) and decreased the transcriptional activity of CREB on SOD2. Taken together, our findings revealed a critical role of IL-13 in the induction of salivary gland epithelial cell senescence through the elevated mitochondrial oxidative stress in a STAT6-CREB-SOD2-dependent pathway in IgG4-RS.
Cellular Senescence/genetics* ; Humans ; Immunoglobulin G/metabolism* ; Interleukin-13/pharmacology* ; Mitochondria/metabolism* ; Sialadenitis/metabolism*

Objective To identify the possibility of IgG Fc binding protein (FCGBP) acting as a prognostic marker of low-grade glioma (LGG) and its correlation with immune infiltration. Methods The expression of FCGBP was analyzed in pan-cancer using The Cancer Genome Atlas (TCGA), Genotypic tissue expression (GTEX), and China Glioma Genome Atlas (CGGA) database. Then, GSE15824 and GSE68848 datasets were selected for further verification. And gene expression Profile Interaction analysis (GEPIA) database and R language were used to analyze the relationship between FCGBP and survival prognosis. Metascape and GSEA were used for functional annotation and enrichment analysis. Finally, the expression of FCGBP gene in LGG immune microenvironment and its correlation with immune cells were analyzed by TIMER database. Results FCGBP was highly expressed in LGG tissues, indicating poor prognosis of LGG patients. Receiver operating characteristic (ROC) curve analysis and COX analysis showed that FCGBP was an independent risk factor for the prognosis of LGG. Moreover, Gene Ontology (GO) demonstrated that FCGBP was involved in cell metabolism, localization, positive, and negative regulation of biological processes, as well as biological adhesion, response to viral and microbial stimulation, and inflammation. GSEA pathway enrichment analysis showed that FCGBP was significantly correlated with Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, Toll-like receptor (TLR) pathway, chemokine pathway, and P53 pathway. In addition, FCGBP expression was positively correlated with the expression of most immune cells in the immune microenvironment of LGG. Conclusion The high expression of FCGBP in LGG is a risk factor for survival and prognosis, and it is positively correlated with the expression of immune cells.
Humans ; Prognosis ; Glioma/genetics* ; China ; Gene Ontology ; Immunoglobulin G ; Tumor Microenvironment ; Cell Adhesion Molecules

The DNA coding for the fusion protein of thromobopoietin mimetic peptide (TMP) and human IgG1 Fc fragment was amplified from recombinant plasmid pET28a/TMPFc, inserted into pPICZalphaA and transformed into Pichia pastoris using electroporation. The recombinants of correct phenotype were identified after screening on MDH and MMH culture medium. The fusion gene was verified with PCR and western blot. MTT method was used to test the activity of TMPFc in promoting the growth of Ba/ F3-mpl cell. The TMPFc with a 64 000 molecular weight was a secretary protein in the system and its expression amounted to 65% of the total protein in the medium supernatant. The TMPFc showed a promotive effect on the growth of Ba/F3-mpl in vitro. A significant portion of the secretary protein existed as dimer, which provided material for studying the dimer in future.
Amino Acid Sequence ; Blotting, Western ; Humans ; Immunoglobulin Fc Fragments ; genetics ; Immunoglobulin G ; genetics ; Molecular Sequence Data ; Pichia ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; Thrombopoietin ; genetics

OBJECTIVETo develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.

METHODSThe heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.

RESULTSThe baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.

CONCLUSIONThe recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Antibodies, Monoclonal ; biosynthesis ; immunology ; Baculoviridae ; genetics ; Hepatitis A virus ; immunology ; Hepatitis Antibodies ; biosynthesis ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; immunology ; Immunoglobulin G ; biosynthesis ; immunology ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Recombinant Proteins ; biosynthesis ; immunology

Streptococcus suis (S. suis) IgG-binding protein (SPG) was present in all S. suis strains examined. It showed binding activities with IgG from various host species. Little was known about the biological role of this protein, but it was commonly believed that it acted as virulence factor. In this study, the genes encoding SPG were amplified respectively from the total DNA of the S. suis serotype 1/2, 1, 2 and 9 with PCR and expressed in Escherichia coli BL21 by plasmid pET28a as vector. The recombinant proteins were first purified with affinity chromatography (Ni-NTA), and further purified by sephadexG-200 gel chromatography. The recombinant SPG proteins were identified to have binding activities with IgG of different host species, and for human and porcine IgG they showed better binding activities. But the SPG from different serotypes of S. suis showed no great differences in their binding activities with IgG from the same host species.
Bacterial Proteins ; genetics ; metabolism ; Binding Sites, Antibody ; genetics ; Escherichia coli ; genetics ; metabolism ; Immunoglobulin G ; immunology ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Streptococcus suis ; immunology

OBJECTIVETo construct a recombinant adenovirus vector carrying soluble extracellular region of tumor necrosis factor alpha receptor I-IgGFc (sTNFRI-IgGFc) and express the fusion protein in human bronchial epithelial HBE135-E6E7 cells.

METHODSsTNFRI-IgGFc fusion gene was subcloned into the adenovirus shuttle plasmid pDC316, which was co-transfected with helper plasmid pBHGloxPE1,3Cre into HEK293 cells. The recombinant adenovirus (Ad-sTNFRI-IgGFc) was generated by homologous recombination of the 2 plasmids in HEK293 cells. After identification with PCR, Ad-sTNFRI-IgGFc was amplified and purified, and its titer measured using TCID50 assay. The transcription and expression of sTNFRI-IgGFc gene in the transfected HBE135-E6E7 were detected by RT-PCR and immunohistochemistry.

RESULTSAd-sTNFRI-IgGFc was successfully constructed with a viral titer of 3 x 10(10) TCID50/ml. The expression of sTNFRI-IgGFc mRNA and protein was confirmed in the transfected HBE135-E6E7 cells.

CONCLUSIONThe constructed Ad-sTNFRI-IgGFc can effectively infect HBE135-E6E7 cells for efficient expression of sTNFRI-IgGFc protein, which antagonizes the cytolytic effect of TNFalpha in L929 cells, suggesting the potential of adenovirus expressing sTNFRI-IgGFc for local treatment of asthma.

Adenoviridae ; genetics ; Bronchi ; cytology ; Epithelial Cells ; cytology ; metabolism ; Genetic Vectors ; genetics ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor, Type I ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

To obtain human antibodies against the Gn protein of Severe fever with thrombocytopenia syndrome virus (SFTSV) with phage display technology, this study aimed to screen anti-Gn protein antibodies from an anti-SFTSV Fab human phage display library. Antibody genes were identified by sequence analysis and the specificity of antibodies was confirmed by ELISA. The Fab antibody genes were cloned into the HL51-14 vector and expressed in a mammalian cell expression system. IgG antibodies were then purified by protein A affinity chromatography,and the results were further confirmed by ELISA,IFA,western blotting assays and micro-neutralization tests. The results showed that, after three rounds of panning, there were 390 human Fab antibodies against SFTSV particles, of which 364 were specific for nucleoprotein. Coated with the Gn protein, eight different Fab antibodies specific for Gn protein were obtained after the determination of the subtype and subclass of antibodies by gene sequencing; five of these antibodies were from the Lambda library and three were from the Kappa library. The eight IgG antibodies could specifically bind to Gn protein according to the ELISA, IFA and Western blotting assays. The micro-neutralization test showed that these eight antibodies had no neutralizing activity,but they could still provide a reference for research in human monoclonal antibodies against SFTSV.
Antibodies ; genetics ; immunology ; Bunyaviridae Infections ; genetics ; immunology ; virology ; Cell Line ; Cloning, Molecular ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin G ; genetics ; immunology ; Neutralization Tests ; Phlebovirus ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

ATR-Fc is a fusion protein consisting of extracellular domain of human anthrax toxin receptor (ATR) and a fragment (hinge, CH2, and CH3 domains) of the Fc of human IgG1. The aim of ATR-Fc expression is to get an antibody-like molecule binding to protective antigen (PA), a component of anthrax toxins, this fusion protein may compete with cell surface receptor for PA binding, and block the transport of lethal factor (LF) and edema factor (EF) into cells, thereby act as an antitoxin to prevent and treat anthrax infection. A DNA fragment encoding N-terminal amino acids 1-227 of ATR and human IgG1 Fc was inserted into the Hind III and Not I sites of pcDNA3.1 to generate the eukaryotic vector pcDNA3.1/ATR-Fc for expression of ATR-Fc fusion protein. Using lipofectine-mediated gene transfer technique, pcDNA3.1/ATR-Fc was transfected into CHO-K1 cells. After selected with G418, a recombinant CHO cell line, ATR-Fc-1D5, whose expression level was about 10 - 15 microg/(10(6) cells x d), was established. The recombinant protein expressed by the ATR-Fc-1D5 cells was purified with protein A chromatography. The experimental results demonstrated a direct and specific interaction between ATR-Fc and PA assessed by ELISA.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

BACKGROUNDTo acquire the recombinant human monoclonal antibodies and IgG to adeno-associated virus type 2 (AAVs-2).

METHODSConstruct and pan human Fab antibody library to AAVs-2 was established from normal volunteer donors by using phage display technology and secreted expression in E.Coli system. The positive Fab clones were selected and characterized through ELISA and immunofluorescent assay, and then the heavy and light chain were sequenced. The gene of light chain and heavy chain Fd fragment of recombinant mAb were inserted into baculovirus expression vector pAC-L-Fc and construct expression vectors of intact IgG, then transfected insert sf-9 cell secreted expression in Baculovirus/Insert system. Immunoprecipitation test was used to detect its recognizing region.

RESULTSOne clone named AAVs-31 showed positive responses in ELISA and IFA, the Fab was composed of gamma chain and kappa chain IgG was positive in ELISA and IFA. The IgG failed to detect nonassembled or denatured capsid proteins, but recognized the AAVs-2 stock from immunoprecipitation test.

CONCLUSIONThe authors isolated a clone of Fab and IgG to adeno-associated virus type 2 by phage display technology, they perhaps recognize an epitope which is formed during capsid assembly.

Amino Acid Sequence ; Animals ; Antibodies, Viral ; genetics ; immunology ; Cell Line ; Dependovirus ; genetics ; immunology ; Gene Expression ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin G ; genetics ; immunology ; Molecular Sequence Data ; Peptide Library ; Recombinant Proteins ; genetics ; immunology ; Spodoptera