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

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

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlandia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlandia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlandia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlandia and Sao Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.
Animals ; Antigens, Bacterial/blood/*diagnostic use ; Bacterial Outer Membrane Proteins/genetics/metabolism ; Bacterial Proteins/*genetics/metabolism ; Base Sequence ; Brazil ; Dog Diseases/diagnosis/*microbiology ; Dogs ; Ehrlichia canis/*genetics/*immunology/isolation & purification ; Ehrlichiosis/diagnosis/microbiology/*veterinary ; Fluorescent Antibody Technique, Indirect/veterinary ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; RNA, Ribosomal, 16S/genetics/metabolism ; Sequence Alignment/veterinary

Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 (PLP2-TM) of coronaviruses acts as a novel autophagy-inducing protein. Intriguingly, PLP2-TM induces incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2-TM interacts with the key autophagy regulators, LC3 and Beclin1, and promotes Beclin1 interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclin1 partially reverses PLP2-TM's inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results suggested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclin1, which in turn modulates coronavirus replication and antiviral innate immunity.
Apoptosis Regulatory Proteins ; antagonists & inhibitors ; genetics ; immunology ; Autophagy ; Beclin-1 ; Coronavirus NL63, Human ; genetics ; immunology ; Gene Expression Regulation ; HEK293 Cells ; HeLa Cells ; Host-Pathogen Interactions ; immunology ; Humans ; Immune Evasion ; Immunity, Innate ; Interferon-gamma ; genetics ; immunology ; Lysosomes ; metabolism ; virology ; MCF-7 Cells ; Membrane Fusion ; Membrane Proteins ; antagonists & inhibitors ; genetics ; immunology ; Microtubule-Associated Proteins ; genetics ; immunology ; Papain ; genetics ; immunology ; Phagosomes ; metabolism ; virology ; RNA, Small Interfering ; genetics ; immunology ; Signal Transduction ; Virus Replication

The aim of this study is to construct a SARS-CoV S protein-specific phage displayed antigen library for the epitope characterization of anti-S monoclonal antibodies (mAbs). First, the full-length gene of SARS-S protein was PCR amplified, purified and then digested with DNase I to obtain DNA fragments in the size range of 50-500 bp. The resulting fragments were blunt-end ligated to the modified phage display vector pComb3XSS. The reactions were electrotransformed into XL1-Blue and infected with VCSM13 helper phage. The SARS-CoV S protein-specific phage displayed antigen library was biopanned and screened against two anti-S mAbs, S-M1 and S-M2. The results showed that we successfully constructed the phage displayed antigen library with a size of 5.7 x 10(6). After three-rounds of biopanning, 14 positive phage clones for S-M1 and 15 for S-M2 were respectively identified. Sequence analyses revealed the possible epitopes of two mAbs. Therefore, the S protein-specific phage displayed antigen library provides a crucial platform for the epitope characterization of anti-S antibodies and it is highly valuable for development of SARS vaccines and diagnostics.
Antibodies, Viral ; immunology ; Bacteriophages ; genetics ; metabolism ; Epitopes ; genetics ; immunology ; Humans ; Membrane Glycoproteins ; genetics ; immunology ; Peptide Library ; SARS Virus ; genetics ; immunology ; Severe Acute Respiratory Syndrome ; immunology ; virology ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; genetics ; immunology

OBJECTIVETo determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism.

METHODSOmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays.

RESULTSThe bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01).

CONCLUSIONExpression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cell Line ; Chaperonin 60 ; genetics ; metabolism ; Humans ; Leptospira interrogans ; genetics ; immunology ; pathogenicity ; Lipoproteins ; genetics ; metabolism ; Macrophages ; microbiology

OBJECTIVETo explore the expression and clinical significance of T cell immunoglobulin mucin (TIM)-1, TIM-3 and T cell-specific transcription factors T-bet and GATA-3 in spleen mononuclear cells in patients with primary immune thrombocytopenia (ITP).

METHODSThe spleen samples were obtained from 17 active ITP patients and 10 controls with spleen traumatic rupture. By using real-time quantitative polymerase chain reaction, the mRNA expressions of TIM-3, TIM1, T-bet and GATA-3 were studied in all subjects.

RESULTSTIM-3 mRNA levels of active ITP patients were significantly decreased to (29 ± 16)% of that of control, TIM-1 mRNA levels of active ITP patients increased to (3.20 ± 2.18) folds of that of control, but the difference was not significant. The ratio of TIM-1/ TIM-3 was elevated in active ITP patients. T-bet mRNA levels were up-regulated in ITP patients by (2.82 ± 1.57) folds (P<0.05) and the expression of GATA3 was decreased by 14% folds (P<0.05) compared to controls. The ratio of T-bet/GATA3 were significantly elevated in ITP patients.

CONCLUSIONThe imbalance between TIM-3 and TIM-1 expression might play an important role in pathogenesis of ITP.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Case-Control Studies ; Female ; Flow Cytometry ; GATA3 Transcription Factor ; metabolism ; Hepatitis A Virus Cellular Receptor 1 ; Hepatitis A Virus Cellular Receptor 2 ; Humans ; Male ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; metabolism ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; metabolism ; RNA, Messenger ; genetics ; Receptors, Virus ; metabolism ; Spleen ; metabolism ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Young Adult

Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell. Amongst the spillage, the secondary messenger molecule, cyclic dimeric guanosine monophosphate (c di-GMP), was recently shown to be bound by stimulator of interferon genes (STING). Binding of c di-GMP by STING activates the Tank Binding Kinase (TBK1) mediated signaling cascades that galvanize the body's defenses for elimination of the pathogen. In addition to c di-GMP, STING has also been shown to function in innate immune responses against pathogen associated molecular patterns (PAMPs) originating from the DNA or RNA of pathogens. The pivotal role of STING in host defense is exemplified by the fact that STING(-/-) mice die upon infection by HSV-1. Thus, STING plays an essential role in innate immune responses against pathogens. This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the immune defenses against invading microbes. Similarly, STING could be targeted for mitigating the inflammatory responses augmented by the innate immune system. This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in delineating the mechanism of STING-mediated responses.
Animals ; Cyclic GMP ; physiology ; Dimerization ; Herpes Simplex ; immunology ; pathology ; Humans ; Immunity, Innate ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Protein Binding ; RNA, Viral ; metabolism ; STAT6 Transcription Factor ; metabolism ; Second Messenger Systems

The p38 mitogen-activated protein kinase (MAPK) plays an evolutionarily conserved role in the cellular response to microbial infection and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. In the Caenorhabditis elegans, the p38 MAPK (also called PMK-1) signaling pathway has been shown to be required in its resistance to bacterial infection. However, how different upstream MAP2Ks and MAP3Ks specifically contribute to the activation of PMK-1 in response to bacterial infection still is not clearly understood. By using double-stranded RNA-mediated interference (RNAi) and genetic mutants of C. elegans, we demonstrate that C. elegans MOM-4, a mammalian TAK1 homolog, is required for the resistance of C. elegans to a P. aeruginosa infection. We have also found that the MKK-4 of C. elegans is required for P. aeruginosa resistance, but not through the regulation of DLK-1. In summary, our results indicate that different upstream MAPKKKs or MAPKKs regulate the activation of PMK-1 in response to P. Aeruginosa.
Animals ; Caenorhabditis elegans ; enzymology ; genetics ; immunology ; microbiology ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; Disease Resistance ; Enzyme Activation ; MAP Kinase Kinase 1 ; metabolism ; MAP Kinase Signaling System ; Membrane Proteins ; deficiency ; genetics ; metabolism ; Mutation ; Pseudomonas Infections ; enzymology ; Pseudomonas aeruginosa ; physiology ; RNA Interference ; p38 Mitogen-Activated Protein Kinases ; metabolism