Objective To investigate the effects of hyaluronic acid and proteoglycan-linked protein 1 (HAPLN1) secreted by synovial fibroblasts (FLS) on the polarization of macrophages (Mϕ) in rheumatoid arthritis (RA). Methods Human monocytic leukemia cells (THP-1) were differentiated into Mϕ, which were subsequently exposed to recombinant HAPLN1 (rHAPLN1). RA-FLS were transfected separately with HAPLN1 overexpression plasmid (HAPLN1^OE) or small interfering RNA targeting HAPLN1 (si-HAPLN1), and then co-cultured with Mϕ to establish a co-culture model. The viability of Mϕ was assessed using the CCK-8 assay, and the proportions of pro-inflammatory M1-type and anti-inflammatory M2-type Mϕ were analyzed by flow cytometry. Additionally, the expression levels of inflammatory markers, including interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS), were quantified using quantitative real-time PCR and Western blot analysis. Results The viability of Mϕ was increased in the rHAPLN1 group compared to the control group. Furthermore, both the M1/Mϕ ratio and inflammatory factor levels were elevated in the rHAPLN1 and HAPLN1^OE groups. In contrast, the si-HAPLN1 group exhibited a decrease in the M1/Mϕ ratio and inflammatory factor expression. Notably, the introduction of rHAPLN1 in rescue experiments further promoted Mϕ polarization towards the M1 phenotype. Conclusion HAPLN1, secreted by RA fibroblast-like synoviocytes (RA-FLS), enhances Mϕ polarization towards the M1 phenotype.
Humans ; Arthritis, Rheumatoid/genetics* ; Macrophages/immunology* ; Fibroblasts/metabolism* ; Phenotype ; Extracellular Matrix Proteins/genetics* ; Proteoglycans/genetics* ; Synovial Membrane/cytology* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics* ; Nitric Oxide Synthase Type II/genetics* ; Cell Differentiation ; Coculture Techniques ; THP-1 Cells

Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

Acinetobacter baumannii (A. Baumannii) is an emerging opportunistic pathogen responsible for hospital-acquired infections, and which now constitutes a sufficiently serious threat to public health to necessitate the development of an effective vaccine. In this study, a recombinant fused protein named OmpK/Omp22 and two individual proteins OmpK and Omp22 were obtained using recombinant expression and Ni-affinity purification. Groups of BALB/c mice were immunized with these proteins and challenged with a clinically isolated strain of A. baumannii. The bacterial load in the blood, pathological changes in the lung tissue and survival rates after challenge were evaluated. Mice immunized with OmpK/Omp22 fused protein provided significantly greater protection against A. baumannii challenge than those immunized with either of the two proteins individually. The results provide novel clues for future design of vaccines against A. baumannii.
Acinetobacter Infections ; pathology ; prevention & control ; Acinetobacter baumannii ; genetics ; immunology ; Animals ; Antibodies, Bacterial ; blood ; Bacterial Load ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Disease Models, Animal ; Female ; Mice, Inbred BALB C ; Pneumonia, Bacterial ; pathology ; prevention & control ; Recombinant Fusion Proteins ; genetics ; immunology

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

OBJECTIVEChlamydia psittaci is an avian respiratory pathogen and zoonotic agent. The wide prevalence of C. psittaci poses a threat to the poultry industry and its employees. However, few commercial kits are available for detecting avian antibodies excluding the in-house ELISA kit. In this study, we developed a novel ELISA kit for detecting antibodies against C. psittaci based on the N-terminal fragment of polymorphic outer membrane protein D (PmpD-N) as the coating antigen.

METHODSThe antigen concentrations, primary antibody, and cut-off value were determined and optimized. The ELISA, designated PmpD-N ELISA, was assessed for sensitivity, specificity, and concordance using sera samples from 48 experimentally infected and 168 uninfected SPF chickens.

RESULTSThe sensitivity and specificity of PmpD-N ELISA were 97.9%, 100%, respectively, while the concordance was 98.1% as compared to that of MOMP-ELISA. No cross-reaction with positive sera for other avian pathogens was found. Using PmpD-N ELISA, 799/836 clinical samples were positive, including 93.0% and 98.1% positivity in layers and broilers, respectively.

CONCLUSIONThese data indicate that indirect ELISA with PmpD-N as the antigen candidate is a promising approach for the surveillance of C. psittaci infection.

Animals ; Bacterial Proteins ; analysis ; Chickens ; Chlamydophila psittaci ; genetics ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; veterinary ; Membrane Proteins ; analysis ; Poultry Diseases ; diagnosis ; microbiology ; Psittacosis ; diagnosis ; microbiology ; veterinary ; Sensitivity and Specificity

Interferon-induced Transmembrane Proteins (IFITMs) were identified through small interference RNA (siRNA) screening method in 1980s. The antiviral properties of the IFITMs were firstly discovered in 1996. Recently, its antiviral effect and mechanism have become a research hotspot. Many studies have shown that IFITM can inhibit the replication of multiple pathogenic viruses, including influenza A virus (IAV), Human Immunodeficiency Virus (HIV-1), hepatitis C virus (HCV), Ebola virus (EBOV), West Nile virus and so on. IFITMs inhibit the replication of virus in the early stage of the viral life cycle, which occurred before the release of viral genomes into the cytosol. Recent studies indicate that IFITM proteins could block viral replication by mediate viral membrane fusion. However, the mechanism is still under investigation. Here we review the discovery and characterization of the IFITM proteins, elucidate their antiviral activities and the potential mechanisms.
Animals ; Humans ; Interferons ; genetics ; immunology ; Membrane Proteins ; genetics ; immunology ; Virus Diseases ; genetics ; immunology ; virology ; Viruses ; genetics ; immunology

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

No abstract available.
Bone Marrow/metabolism/pathology ; DNA Mutational Analysis ; Gene Rearrangement, T-Lymphocyte ; Humans ; Immunophenotyping ; Infant ; Lymphohistiocytosis, Hemophagocytic/*diagnosis ; Male ; Membrane Proteins/chemistry/genetics ; Polymorphism, Single-Stranded Conformational ; Republic of Korea ; T-Lymphocytes/immunology/*metabolism

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by abnormal proliferation of synoviocytes, leukocyte infiltration, and angiogenesis. The endoplasmic reticulum (ER) is the site of biosynthesis for all secreted and membrane proteins. The accumulation of unfolded proteins in the ER leads to a condition known as ER stress. Failure of the ER's adaptive capacity results in abnormal activation of the unfolded protein response. Recently, we have demonstrated that ER stress-associated gene signatures are highly expressed in RA synovium and synovial cells. Mice with Grp78 haploinsufficiency exhibit the suppression of experimentally induced arthritis, suggesting that the ER chaperone GRP78 is crucial for RA pathogenesis. Moreover, increasing evidence has suggested that GRP78 participates in antibody generation, T cell proliferation, and pro-inflammatory cytokine production, and is therefore one of the potential therapeutic targets for RA. In this review, we discuss the putative, pathophysiological roles of ER stress and GRP78 in RA pathogenesis.
Animals ; Arthritis, Rheumatoid/genetics/*pathology ; Autoantibodies/immunology ; Cell Proliferation ; Cytokines/biosynthesis/immunology ; Endoplasmic Reticulum/immunology/pathology ; Endoplasmic Reticulum Stress/*immunology ; Haploinsufficiency/genetics ; Heat-Shock Proteins/*genetics/*immunology ; Humans ; Lymphocyte Activation ; Mice ; Neovascularization, Pathologic/genetics ; Protein Folding ; Synovial Membrane/cytology ; T-Lymphocytes/immunology ; Unfolded Protein Response/*immunology

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.
Antibodies, Protozoan/*blood ; Antigens, Protozoan/diagnostic use/*genetics ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Membrane Glycoproteins/*genetics ; Protozoan Proteins/*genetics ; Recombinant Proteins/diagnostic use/immunology ; Sensitivity and Specificity ; Toxoplasma/immunology ; Toxoplasmosis/blood/*diagnosis