A20 was originally identified as a TNFα-induced protein 3 (TNFAIP3), a key regulator of inflammation signalling pathways, as well as a NF-κB inhibitor. It plays a critical role in regulation of innate and adoptive immunity. Recently, A20 has also been proposed to function as a tumor suppressor. Lacking A20 gene is involved in inflammation-mediated autoimmune disease and tumorigenesis in several human B-cell lymphomas. Current advance concerning the feature of A20 expression in immune cells, the biological function, the immune regulated function in native immunity, humoral and cellular immunity, the inactivation of A20 in lymphocytic malignancies and the polymorphism and abnormal expression of A20 in autoimmune disease indicate that the clinical significance of A20 should be worthy to recognize and to be further employed in induction of immune tolerance, antitumor immuno-regulated therapy and antiviral immunotherapy and so on.
Adaptive Immunity ; Autoimmune Diseases ; immunology ; DNA-Binding Proteins ; immunology ; Genes, Tumor Suppressor ; Humans ; Inflammation ; immunology ; Intracellular Signaling Peptides and Proteins ; immunology ; NF-kappa B ; immunology ; Nuclear Proteins ; immunology ; Tumor Necrosis Factor alpha-Induced Protein 3 ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo express human HZF1 fusion protein in E. coli and to obtain an anti-HZF1 antibody.

METHODSA DNA fragment encoding non-zinc finger region of HZF1 protein was inserted into pET30a vector to get the recombination expression plasmid pET30a-HZF1. E. coli was transformed with pET30a-HZF1 and the selected clones were cultured with isopropy-beta-D-thiogalactoside induction. The proteins were prepared from the culture and the fusion protein was purified by Ni column. Rabbits were immunized and reinforced three times with the purified fusion protein. The antiserum was collected and the titer and the specificity of the antibody were checked by ELISA and Western blot.

RESULTSAntibody against HZF1 was obtained and its titer was more than 1:100 000, as proven by ELISA. Western blot analysis showed specific reaction between this antibody and HZF1 fusion protein or the endogenetic HZF1 protein in hemin-induced K562 cells.

CONCLUSIONSThe specific antibody against HZF1 is obtained. The antibody may have potential application in farther HZF1 function study and HZF1 determination in tissues and cells.

DNA-Binding Proteins ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Transfer Techniques ; Immune Sera ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Transformation, Bacterial

Animals ; Antibodies, Bacterial ; blood ; Bacterial Proteins ; genetics ; immunology ; Brucella Vaccine ; immunology ; Brucella abortus ; immunology ; Female ; Immunization ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Periplasmic Binding Proteins ; genetics ; immunology ; Ribosomal Proteins ; genetics ; immunology ; Vaccines, DNA ; immunology

Streptococcus mutans (S. mutans), a major aetiologic agent of dental caries, is involved in systemic diseases, such as bacterial endocarditis, if it enters the bloodstream through temporary bacteraemia. Interleukin (IL)-1β, a proinflammatory cytokine, is related to the host defences against pathogens, and its synthesis, maturation, and secretion are tightly regulated by the activation of the inflammasome, an inflammatory signalling complex. This study examined the signalling mechanism of IL-1β secretion and the inflammasome pathway induced by S. mutans to explain the molecular mechanism through which systemic infection by oral streptococci can occur. After infection of THP-1 cells with S. mutans, the expression of inflammasome components was detected using various methods. S. mutans induced IL-1β secretion via caspase-1 activation, and S. mutans-induced IL-1β secretion required absent in melanoma (AIM2), NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasome activation. In particular, the S. mutans-induced NLRP3 inflammasome was mediated by adenosine triphosphate (ATP) release, potassium depletion and lysosomal damage. Our study provides novel insight into the innate immune response against S. mutans infection.
Blotting, Western ; CARD Signaling Adaptor Proteins ; immunology ; Calcium-Binding Proteins ; immunology ; Caspase 1 ; immunology ; DNA-Binding Proteins ; immunology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunity, Innate ; Inflammasomes ; immunology ; Interleukin-1beta ; immunology ; Macrophages ; immunology ; NLR Family, Pyrin Domain-Containing 3 Protein ; immunology ; Signal Transduction ; Streptococcus mutans ; immunology ; Tumor Necrosis Factor-alpha ; immunology

High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA.
Antibodies, Antinuclear* ; DNA/immunology ; DNA/analysis* ; DNA-Binding Proteins/chemistry ; Gold ; Membranes, Artificial ; Polymerase Chain Reaction ; Polyvinyls/chemistry ; Radioimmunoassay/methods* ; Thioctic Acid/chemistry

Knull phenotype completely lacks all Kell system antigens. Anti-Ku antibody is seen in immunized persons with Knull phenotype by transfusion or pregnancy. It can cause a fatal hemolytic transfusion reaction. A 66-yr-old male patient with liver cirrhosis visited emergency center due to acute bleeding. The patient was at hypovolemic shock status: his blood pressure was 80/50 mmHg, pulse rate was 110/min and hemoglobin level was 4.4 g/dL. Because of the presence of antibody against high incidence antigen, we could not find any compatible blood for the patient. Nevertheless, 4 units of packed RBCs had to be transfused. Moderate hemolytic transfusion reaction was developed after transfusion. At endoscopic examination, blood was spurting from gastric cardiac varix. Endoscopic histoacryl injection was tried, and bleeding was successfully controlled. After bleeding stopped, he was managed for anemia using steroid and other medical therapy instead of transfusion. His hemoglobin level was improved to 7.7 g/dL at the time of discharge. Later he has been proved to have a Knull phenotype, which is very rare, and anti-Ku antibody. This report is the first case of anti-Ku in a Knull phenotype person in Korea, who experienced a moderate hemolytic transfusion reaction.
Aged ; Antigens, Nuclear/*immunology ; Blood Group Incompatibility ; Blood Transfusion/*adverse effects ; DNA-Binding Proteins/*immunology ; Humans ; Isoantibodies/blood ; Kell Blood-Group System/*genetics ; Korea ; Male ; Phenotype

The host takes use of pattern recognition receptors (PRRs) to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.
DNA, Bacterial ; immunology ; metabolism ; DNA, Viral ; immunology ; metabolism ; Gene Expression Regulation ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Interferon Regulatory Factor-3 ; genetics ; immunology ; Interferon Type I ; biosynthesis ; immunology ; Membrane Proteins ; genetics ; immunology ; Models, Molecular ; NF-kappa B ; genetics ; immunology ; Nucleotides, Cyclic ; biosynthesis ; immunology ; Nucleotidyltransferases ; genetics ; immunology ; Protein Binding ; Protein-Serine-Threonine Kinases ; genetics ; immunology ; Signal Transduction

We studied the immunogenicity of pseudorabies virus gC DNA vaccination by fusing the murine complement C3d receptor binding domain. First, pseudorabies virus gC gene was linked to four copies of C3d receptor binding domain (M284), and then cloned into the vector pcDNA3.1 to construct the recombinant plasmid sgC-M284. Through the experiment of immunized BALB/c mice, we found that the enzyme linked immunosorbent assay (ELISA) antibody titer for sgC-M284 was 17-fold higher than that for sgC alone, and protective rate of mice was augmented from 25% to 88% after lethal dose PrV (316 LD50) challenge. In addition, the IL-4 levels for sgC-M284 immunization approached that for the pseudorabies virus inactivated vaccine. In conclusion, we demonstrated murine C3d receptor binding domain fusion significantly increased Th2-biased immune response by inducing IL-4 production.
Adjuvants, Immunologic ; physiology ; Animals ; Antibody Formation ; immunology ; Binding Sites ; Cloning, Molecular ; Complement C3d ; genetics ; immunology ; Herpesvirus 1, Suid ; genetics ; immunology ; Interleukin-4 ; immunology ; Mice ; Mice, Inbred BALB C ; Pseudorabies Vaccines ; immunology ; Receptors, Complement 3d ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; pharmacology ; Viral Fusion Proteins ; immunology

Autoimmune sera have been used in the diagnosis of autoimmune diseases as well as the analysis of nuclear substructures. In an attempt to study the biological characteristics of the nuclear matrix, we screened human sera using immunofluorescent staining and immunoblot. We detected antibodies against nuclear matrix (NM), a remnant nonchromatin protein compartment after the treatment of detergent, salt and nuclease, in 212 out of 284 tested sera (74.6%) by immunoblot. Peptides with molecular weights of 70 kDa, 50 kDa and 25 kDa were detected in the order of frequency. Clinical informations of 198 out of 212 cases were available and went as follows: 38 cases were autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; 132 non-autoimmune and non-neoplastic diseases; 16 neoplastic diseases and 12 cases unclassified. The immunofluorescent staining intensity by anti-nuclear matrix protein (NMP) antibodies decreased variably, but fibrillogranular, speckled and nucleolar immunolocalization patterns were retained after in situ fractionation. Ku70 and La protein were detected by anti-NMP antibodies. Immunolocalization by anti-NMP antibodies indicates that the NMPs constitute a variety of characteristic nuclear substructures and may serve as autoantigens in diverse human diseases. In addition, the presence of Ku70 and La protein as NMPs suggests that the NM can be functionally active in association with DNA or RNA.
Autoantigens/analysis* ; Autoimmune Diseases/immunology ; Autoimmune Diseases/blood ; Base Sequence ; DNA, Complementary ; DNA-Binding Proteins/analysis* ; Fluorescent Antibody Technique, Indirect ; Hela Cells ; Human ; Immunoblotting ; Molecular Sequence Data ; Nuclear Matrix/immunology ; Nuclear Proteins/analysis* ; Ribonucleoproteins/analysis* ; Tumor Cells, Cultured

Tissue and organ differentiation is tightly controlled to ensure proper development and function of the growing embryo as well as cells such as lymphocytes that differentiate throughout the adult stage. Therefore it is vital that the genes and the protein they encode that are involved in these processes function accurately. Hence, any mutation or error that occurs along the way can result in extensive damage, which is expressed in various ways in the embryo and can result in immune pathogenesis, including immunodeficiency and autoimmune diseases, when lymphocyte development is altered. A number of studies have been carried out to look at the genes regulating transcription in tissue differentiation, including the transcription factors Pbx1. This gene is of particular interest to us as we have identified that it is associated with systemic lupus erythematosus susceptibility (Cuda et al., in press). This perspective summarizes the known roles of Pbx1 in tissue differentiation as well as our recent findings associating genetic variations in Pbx1 to lupus susceptibility, and we will speculate on how this gene controls the maintenance of immune tolerance in T cells.
Animals ; Cell Differentiation ; Chromatin Immunoprecipitation ; DNA-Binding Proteins ; genetics ; immunology ; Genetic Loci ; immunology ; Genetic Predisposition to Disease ; Homeodomain Proteins ; genetics ; immunology ; Humans ; Immune Tolerance ; Lupus Erythematosus, Systemic ; genetics ; immunology ; Lymphocyte Activation ; Mice ; Mice, Transgenic ; Pre-B-Cell Leukemia Transcription Factor 1 ; Protein Structure, Tertiary ; Proto-Oncogene Proteins ; genetics ; immunology ; Signal Transduction ; T-Lymphocytes, Regulatory ; cytology ; immunology ; Transcription Factors ; genetics ; immunology ; Tretinoin ; metabolism