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ABSTRACT: Mycobacterium bovis parasitizes host macrophages and has developed strategies to survive within macrophages. Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence. To further elucidate the molecular pathogenesis of tuberculosis and host response to M. bovis, we explored the mechanisms by which PE_PGRS62 protein increase persistence of mycobacterium within host macrophages. We found that the M. smegmatis strain expressing M. bovis PE_PGRS 62 protein reduced phagolysosome maturation in human macrophages, and significantly decreased the mRNA expression of IL-1β in a dose- and time-dependent. We identified that IFN-γ priming of macrophages immediately prior to infection with PE_PGRS62 expressing M. smegmantis, enhanced the maturation of phagolysosomes and induced IL-1β production both that the protein and mRNA levels and further activated the NF-κB pathway. Overall, we demonstrated that PE_PGRS62 protein altered the immune environment of the host cells, which suggested that the pathogenic PE_PGRS62 protein altering the immune mechanism might be involved in the pathogenesis of mycobacterial disease and hence influenced host cell responses to M. bovis infection.
Veterinary Microbiology 05/2012; 160(1-2):117-25. · 3.33 Impact Factor
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ABSTRACT: IgG was traditionally thought to neutralize virions by blocking their attachment to or penetration into mucosal epithelial cells, a common site of exposure to viruses. However, we describe an intracellular neutralizing action for an influenza hemagglutinin-specific monoclonal antibody, Y8-10C2 (Y8), which has neutralizing activity only at an acidic pH. When Y8 was applied to the basolateral surface of Madin-Darby canine kidney cells expressing the rat neonatal Fc receptor for IgG (FcRn), it significantly reduced viral replication following apical exposure of the cell monolayer to influenza virus. Virus neutralization by Y8 mAb was dependent on FcRn expression and its transport of IgG. As both FcRn and Y8 mAb bind their partners only at acidic pH, the Y8 mAb is proposed to carry out its antiviral activity intracellularly. Furthermore, the virus, Y8 mAb, and FcRn colocalized within endosomes, possibly inhibiting the fusion of viral envelopes with endosomal membranes during primary uncoating, and preventing the accumulation of the neutralized viral nucleoprotein antigen in the nucleus. Prophylactic administration of Y8 mAb before viral challenge in WT mice, but not FcRn-KO mice, conferred protection from lethality, prevented weight loss, resulted in a significant reduction in pulmonary virus titers, and largely reduced virus-induced lung pathology. Thus, this study reveals an intracellular mechanism for viral neutralization in polarized epithelial cells that is dependent on FcRn-mediated transport of neutralizing IgG.
Proceedings of the National Academy of Sciences 11/2011; 108(45):18406-11. · 9.68 Impact Factor
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ABSTRACT: Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses. A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces, FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of a Gag-Fc/CpG vaccine protected at a distal mucosal site. Our data suggest that targeting of FcRn with chimeric immunogens may be an important strategy for mucosal immunization and should be considered a new approach for preventive HIV vaccines.
Journal of Virology 08/2011; 85(20):10542-53. · 5.40 Impact Factor
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ABSTRACT: Almost all infectious diseases are initiated at mucosal surfaces, yet intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal activation of the mucosal immune system. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. We mimicked this process by fusing a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD, to an IgG Fc fragment. Intranasal immunization, together with the adjuvant CpG, completely protected wild-type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B- and T-cell immune responses, with stable protection for at least 6 months after vaccination in most of the immunized animals. The FcRn-IgG transcellular transport pathway may provide a general delivery route for subunit vaccines against many mucosal pathogens.
Nature Biotechnology 02/2011; 29(2):158-63. · 29.50 Impact Factor
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ABSTRACT: PrPC (cellular prion protein) is a GPI (glycophosphatidylinositol)-anchored protein present on the surface of a number of peripheral blood cells. PrPC must be present for the generation and propagation of pathogenic conformer [PrPSc (scrapie prion protein)], which is a conformational conversion form of PrPC and has a central role in transmissible spongiform encephalopathies. It is important to determine the transportation mechanism of normal PrPC between cells. Exosomes are membrane vesicles released into the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. We have identified that THP-1 monocytes can secrete exosomes to culture medium, and the secreted exosomes can bear PrPC. We also found that Hsp70 interacts with PrPC not only in intracellular environment, but in the secreted exosomes. However, the specific markers of exosomes, Tsg101 and flotillin-1, were found with no interaction with PrPC. Our results demonstrated that PrPC can be released from THP-1 monocytes via secreted exosomes, and in this process, Hsp70 binds to PrPC, which suggests that Hsp70 may play a potential functional role in the release of PrPC.
Cell Biology International 10/2010; 35(6):553-8. · 1.48 Impact Factor
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ABSTRACT: Prion diseases are infectious and fatal neurodegenerative disorders. The cellular prion protein (PrP(C)) converting into misfolded isoform of prion protein (PrP(Sc)) is responsible for prion disease infection. Immune system plays an important role in facilitating the spread of prion infections from the periphery to the central nervous system. Macrophages were considered associated with the transportation and replication of PrP(Sc). So, understanding the PrP(C) trafficking in macrophages is important to explore the transport mechanism for PrP(Sc). Here, we isolated exosomes from the culture medium of Ana-1 macrophage cell line and investigated the PrP(C) trafficked by exosomes and the interaction of PrP(C) with Hsp70 in secreted exosomes by western blotting, immunoelectron microscopy, and co-immunoprecipitation. The results showed that the isolated vesicles from the culture medium of macrophages were characterized by exosomes and bore PrP(C). And PrP(C) bound to Hsp70 both in intracellular environment and secreted exosomes. In contrast, PrP(C) had no interaction with marker proteins of exosomes, Tag101 and Flotillin-1. These results suggested that PrP(C) present in extracellular space might be externalized through secreted exosomes from macrophages, and Hsp70 may play roles in the process of PrP(C) released via secreted exosomes.
Acta Biochimica et Biophysica Sinica 05/2010; 42(5):345-50. · 1.38 Impact Factor
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ABSTRACT: The pathogenesis of tuberculosis causing Mycobacterium bovis is largely due to its successful entry and survival in macrophages. Previous research indicated that mycobacteria-specific PE_PGRS genes code for cell surface proteins which may have role in mediating interactions with macrophages. In this study, we expressed PE_PGRS 62 gene in a non-pathogenic fast growing Mycobacterium smegmatis strain and found that the recombinant Mycobacterium smegmatis decreased macrophages livability in a dosage-dependent manner and time-dependent manner, compared with parental strain containing the vector only. To explore whether PE_PGRS 62 modulates the gene expression profile of macrophages, we stimulated macrophages by the M. smegmatis strain expressing PE_PGRS 62 as well as the control strains, followed by real-time RT-PCR assay for the mRNA expression level of IL-1beta, IL-6, and iNOS. The results showed that the expression of IL-1beta, IL-6 in macrophages were down-regulated by stimulation with the M. smegmatis strain expressing PE_PGRS 62 compared to the control strains (P < 0.05). In contrast, there were no measurable differences in the expression of iNOS. Overall, we demonstrated that PE_PGRS 62 protein altered the immune environment of the host cells, which suggest that the pathogenic PE_PGRS 62 protein altering the immune mechanism maybe involved in the pathogenesis of mycobacterial disease.
Molecular and Cellular Biochemistry 03/2010; 340(1-2):223-9. · 2.06 Impact Factor
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ABSTRACT: Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine IFN-gamma. However, in this study we found that IFN-gamma down-regulated the constitutive expression of the neonatal Fc receptor (FcRn), an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation. Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-gamma resulted in a significant decrease of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1 bound to an IFN-gamma activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-gamma, whereas expression of JAK1 or STAT-1 protein restored the IFN-gamma inhibitory effect on luciferase activity. The repressive effect of IFN-gamma on the FcRn promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-gamma activation site sequence and by overexpression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues. Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding protein/p300. Functionally, IFN-gamma stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-regulation of FcRn gene expression by IFN-gamma.
The Journal of Immunology 08/2008; 181(1):449-63. · 5.79 Impact Factor
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ABSTRACT: Neuronal death is a pathological hallmark of prion diseases. Synthetic prion peptide PrP106-126 can convert PrP(C) into protease-resistant aggregates, which can cause neurotoxicity in vivo and in vitro. Various cell surface proteins can participate in the infection process of prions. p75(NTR) can interact with PrP106-126 and has a neurotoxic effect on neurons. However, for p75(NTR) lacking intrinsic catalytic activity domain in cytoplasm, p75(NTR) -associated signaling molecular and the signaling events in cytoplasm in p75(NTR)-mediated apoptosis responding to PrP106-126 remain still unknown. Thus p75(NTR) -associated NF-kappaB signaling pathway was investigated in this study. Herein PrP106-126-induced apoptosis in mouse neuroblastoma cell line N2a, PrP106-126 significantly up-regulated p75(NTR) expression on mRNA and protein levels. For the first time we found that PrP106-126 induced activation of NF-kappaB by Western blot assay, and blocking the interaction of p75(NTR) with PrP106-126 by p75(NTR) polyclonal antibody sc-6189 or pretreatment with inhibitor NF-kappaB SN50 reduced the activation of NF-kappaB and attenuated the apoptotic effect by PrP106-126. This study offers a possible interpretation that NF-kappaB signaling pathway was activated by the interaction of PrP106-126 with p75(NTR), and NF-kappaB activity showed the pro-apoptotic effect in PrP106-126-induced apoptosis in N2a cells. Involvement of NF-kappaB signaling pathway in p75(NTR)-mediated apoptosis may partially account for the PrP106-126-induced neurotoxicity in N2a cells.
Neuroscience Research 06/2008; 62(1):9-14. · 2.25 Impact Factor
