12 research outputs found

    Lipopolysaccharides may aggravate apoptosis through accumulation of autophagosomes in alveolar macrophages of human silicosis

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    <p>Silica dust mainly attacks alveolar macrophages (AMs) and increases the apoptosis of AMs in silicosis patients. However, it is still unclear whether autophagy is affected. Autophagy mainly has defensive functions in response to stress, contributing to cell survival in adverse conditions, and conversely it has also been implicated in cell death. Lipopolysaccharide (LPS) induces autophagy and apoptosis in macrophages. The role of LPS in autophagy and apoptosis in AMs of silicosis patients is unknown. In this study, we collected AMs from 53 male workers exposed to silica and divided them into an observer (control) group, and stage I, II and III patient groups. We found increased levels of LC3B, SQSTM1/p62 and BECN1,whereas the phosphorylation of MTOR,and levels of LAMP2, TLR4, MYD88, TICAM1, as well as the number of lysosomes decreased with the development of silicosis. LPS stimulation triggered autophagy and increased levels of SQSTM1 in AMs. The autophagy inhibitor, 3-methyladenine (3MA), inhibited LPS-induced apoptosis in the AMs of silicosis patients. Moreover, 3MA reversed the LPS-induced decrease in BCL2 and the increase in BAX and CASP3 levels in AMs. These results suggest that autophagosomes accumulate in AMs during silicosis progression. LPS can induce the formation of autophagosomes through a TLR4-dependent pathway, and LPS may exacerbate the apoptosis in AMs. Blockade of the formation of autophagosomes may inhibit LPS-induced apoptosis via the intrinsic apoptotic pathway in AMs. These findings describe novel mechanisms that may lead to new preventive and therapeutic strategies for pulmonary fibrosis.</p

    The role of IL-10 and IFN-γ in the generation and function of the CD8NKT-like cells

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    Cytokine levels in the cell culture media. Cultured CD8NKT-like cells and freshly isolated CD4CD25Treg cells were stimulated with anti-CD3 (1.5 μg/ml) and splenic APCs. At 72 h of culturing, the culture supernatant was saved and used for measuring IL-10, IL-4 and IFN-γ using ELISA. Results are representative of two independent experiments. Suppression activity of CD8NKT-like cells cultured from IFN-γand IL-10mice. CD8NKT-like cells (Tr) cultured from knockout mice and wild-type B6 (WT) mice were co-cultured with naïve CD4CD25responder T cells (Tn) at different Tr/Tn ratios in the presence of splenic APCs and anti-CD3. The cultures were pulsed with 1 μCi/well of [H]thymidine at 72 h and proliferation (cpm) was measured by [H]thymidine incorporation in the last 16 h. Results are expressed as the mean of triplicate cultures. ANOVA -values are <p><b>Copyright information:</b></p><p>Taken from "The IL-10 and IFN-γ pathways are essential to the potent immunosuppressive activity of cultured CD8NKT-like cells"</p><p>http://genomebiology.com/2008/9/7/R119</p><p>Genome Biology 2008;9(7):R119-R119.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2530876.</p><p></p

    Heat map for genes differentially expressed among the four groups of T cells

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    Only those genes with a FDR (q) ≤0.01 and fold change ≥5 are included in this map. Data for each gene are standardized separately before being plotted, as is standard in drawing heat maps, so that all genes have a similar scale and the relative differences for all genes can be visualized on a single plot.<p><b>Copyright information:</b></p><p>Taken from "The IL-10 and IFN-γ pathways are essential to the potent immunosuppressive activity of cultured CD8NKT-like cells"</p><p>http://genomebiology.com/2008/9/7/R119</p><p>Genome Biology 2008;9(7):R119-R119.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2530876.</p><p></p

    Molecular network for the highly upregulated immunity and defense genes

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    The network was created by extracting the direct interactions between these genes from the literature. Three types of relationship are shown in the pathway, binding, expression and regulation. Binding refers to physical interactions between molecules. Expression indicates that the regulator changes the protein level of the target by means of regulating its gene expression or protein stability. Regulation indicates that the regulator changes the activity of the target; the mechanism of the regulation is either unknown or has not been specified in the sentence describing the relationship. This network highlights the importance of two key nodes, IFN-γ and IL-10, which regulate many genes in this network. These genes are also critical for the immunosuppressive function of the CD8 NKT-like cells.<p><b>Copyright information:</b></p><p>Taken from "The IL-10 and IFN-γ pathways are essential to the potent immunosuppressive activity of cultured CD8NKT-like cells"</p><p>http://genomebiology.com/2008/9/7/R119</p><p>Genome Biology 2008;9(7):R119-R119.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2530876.</p><p></p

    Subcellular localization of viral proteins in PK15 cells coinfected with PCV2 and CSFV.

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    <p>PK15-CSFV cells were seeded on glass bottom dishes and inoculated with PCV2 at MOI = 10. At indicated time points after PCV2 infection, cells were fixed and immunostained for PCV2 Cap (green) and CSFV E2 protein (red) as well as DAPI-stained for the nucleus (blue) and observed using CLSM and super-resolution microscopy. The red bar in the merged image represents 10 μm. (A) Overview of PCV2 infection at 72 hpi in PK15-CSFV and ST-CSFV cells. White arrows indicate double-stained cells. (B) Dynamic subcellular localization of viral proteins of PCV2 and CSFV in PK15-CSFV cells. (C) Super-resolution microscopy of PCV2 and CSFV in cells. Cells were fixed and immunostained for N-STORM. Images were taken and reconstructed to obtain 3D models of colocalization of PCV2 Cap and CSFV E2 proteins in the nucleus (white arrows) and in the cytoplasm (blue arrows).</p

    PCV2-induced apoptosis.

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    <p>(A) Cells infected with PCV2 (MOI = 1) were fixed for the TUNEL assay at 72 hpi and immunostained for viral proteins. Apoptotic cells (green), PCV2 Cap proteins (red), CSFV E2 proteins (blue) and nuclei (grey) are shown. (B) Statistical analysis of PCV2-induced apoptosis. Total numbers of TUNEL-positive, PCV2-positive and TUNEL-PCV2 dual-positive cells were counted, and proportions of each type of cells in all labeled (TUNEL or immunostained) cells were calculated in four random fields. (C and D) Cells inoculated with PCV2 of different MOIs were determined by TUNEL assay (C) and measurement of luminescent caspase–3/7 activities (D). The red bar in the merged image indicates 10 μm. Data are represented as means ± SD (n = 3 or more; ns, <i>P</i> > 0.05).</p

    Viral infection status of CSFV in PK15 cell line harboring replicating CSFV.

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    <p>PK15 cells were infected with CSFV and then serially subcultured. Different passages of PK15-CSFV harboring replicating CSFV were collected to detect the viral infection status. (A) Histogram and proportion of CSFV-positive cells by flow cytometry. The red line indicates the positive gate for the cell population. (B) Three indexes of viral infection of PK15-CSFV cells: proportion of CSFV-positive cells using flow cytometry, viral genomic copies in virus stocks by absolute quantitative real-time PCR and infectivity of virus stocks by measurement of TCID<sub>50</sub>. Data are represented as means ± SD (n = 3).</p

    Replication of PCV2 and CSFV in PK15-CSFV cells.

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    <p>PK15 and PK15-CSFV cells were inoculated with PCV2 at MOI = 0.5, 1, 4, 7, 10 and 15. At 72 hpi, cells and supernatants were freeze-thawed to obtain virus stocks. Titers were determined by measurement of TCID<sub>50</sub> in PK15 or ST cells and by absolute quantitative real-time PCR. (A) TCID<sub>50</sub> of PCV2. (B) Genomic copies of PCV2. (C) TCID<sub>50</sub> of CSFV. (D) Genomic copies of CSFV. (E) Total RNA of cells was extracted, and relative quantitative real-time PCR was used to detect the replication level of CSFV in PK15-CSFV cells infected with PCV2 at different MOIs. The ratio of CSFV mRNA to β-actin mRNA in mock-infected PK15-CSFV was defined as 1, and then the relative CSFV mRNA ratio in PCV2-infected PK15-CSFV cells was determined. Data are represented as means ± SD (n = 3; ns, <i>P</i> > 0.05; *<i>P</i> < 0.05; **<i>P</i> < 0.01).</p

    Biological characteristics of PK15-CSFV cells.

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    <p>PK15-CSFV cells were seeded into 6- or 96-well plates and cultured for indicated time points to detect the virus titer, cell viability and apoptosis. (A) Growth curve of CSFV in PK15-CSFV cells. (B) Viability of PK15-CSFV cells by CCK–8. (C) Proportion of TUNEL-positive cells at 72 h. Data are represented as means ± SD (n = 3 or 5; ns, <i>P</i> > 0.05; *<i>P</i> < 0.05; **<i>P</i> < 0.01).</p
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