Journal:

Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

doi: 10.1128/IAI.00004-09

Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P < 0.002 compared to control (Mann-Whitney U test, n = 6).

Article Snippet: For specificity control, some supernatants were preincubated with either 50 μg/ml LPS ( Salmonella enterica serovar Minnesota; Sigma), which has a high binding activity for TLR4/MD-2, or 10 μg/ml anti-mouse TLR4/MD-2 MTS510 MAb (Hycult Biotechnology, Uden, The Netherlands) for 1 h at room temperature.

Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The FASEB Journal

Article Title: Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 v ia TNFα and IFNβ

doi: 10.1096/fj.13-245126

Figure Lengend Snippet: PKR-dependent modulation of Δ isp 2/ isp 3 L. major survival requires TLR2, CD11b, and TLR4. RAW-Bla ( A , B ) or RAW-DN-PKR ( C , D ) cells were infected with Δ isp 2/ isp 3 ( A , C ) or WT L. major ( B , D ) for 3 h, washed and fixed (3 h), or washed and cultured overnight (24 h). Where indicated, cultures were preincubated with 5 μg/ml of antibodies to CD11b, TLR4, TLR2, or control rat IgG2a for 30 min and washed before the addition of parasites. Experiments were performed 3 independent times. Graphs are representative of 1 experiment and indicate means ± sd . * P < 0.0001, ** P < 0.05.

Article Snippet: Anti-mouse CD11b mAbs (clone M1/70), anti-mouse TLR4 neutralizing mAbs (clone MTS510), anti-mouse TLR2 (T2.5), or rat IgG2b (BD Biosciences, San Jose, CA, USA) was incubated at 10 or 5 μg/ml in DMEM/10% FCS for 30 min and removed by extensive washing before the addition of the parasites in DMEM-BSA.

Techniques: Infection, Cell Culture

Journal: The FASEB Journal

Article Title: Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 v ia TNFα and IFNβ

doi: 10.1096/fj.13-245126

Figure Lengend Snippet: TLR2 and TLR4 are recruited to the site of entry of Δ isp 2/ isp 3 L. major in macrophages, and parasite killing requires MyD88 and TRIF. A , B ) Macrophages of C57B6 mice were infected with WT ( A ) or Δ isp 2/ isp 3 L. major ( B ) for 1 h and processed for immunofluorescence, with anti-TLR2 (red) or anti-TLR4 (green). Samples were analyzed by confocal microscopy, sectioned from bottom to top at 0.9 μm, 8 times; images show section 3. Scale bars = 5 μm. Arrows indicate the parasite. C–F ) Macrophages from TLR2-knockout mice ( C ), MyD88-knockout mice ( D ), 129Sv mice ( E ), or TRIF-knockout mice ( F ) were infected for 3 h, washed, and fixed or cultivated for a further 24 h. Experiments were performed 2 separate times. * P < 0.001.

Article Snippet: Anti-mouse CD11b mAbs (clone M1/70), anti-mouse TLR4 neutralizing mAbs (clone MTS510), anti-mouse TLR2 (T2.5), or rat IgG2b (BD Biosciences, San Jose, CA, USA) was incubated at 10 or 5 μg/ml in DMEM/10% FCS for 30 min and removed by extensive washing before the addition of the parasites in DMEM-BSA.

Techniques: Infection, Immunofluorescence, Confocal Microscopy, Knock-Out

Journal: The FASEB Journal

Article Title: Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 v ia TNFα and IFNβ

doi: 10.1096/fj.13-245126

Figure Lengend Snippet: Molecular interactions in macrophages infected with L. major . Contrary to L. amazonensis , PKR activation leads to the killing of L. major , and ISP2 is a factor that prevents PKR activation. In the absence of ISP2, TLR4 and TLR2 are recruited to the site of parasite entry, and proteolytically active NE triggers those receptors ( 1 ), leading to the phosphorylation ( 2 ). The nuclear levels of the NF-κB subunit p65 ( 3a ) and of interferon regulatory factor 3 (IRF3; ( 3b ) are reduced, in comparison, with macrophages infected with WT L. major . The production of TNF-α ( 4 ) and IFNβ ( 5 ) is induced, which results in parasite killing ( 6 ). Activation of TLRs via MyD88 and TRIF is necessary for the killing of ∼50% of the internalized parasites within 24 h.

Article Snippet: Anti-mouse CD11b mAbs (clone M1/70), anti-mouse TLR4 neutralizing mAbs (clone MTS510), anti-mouse TLR2 (T2.5), or rat IgG2b (BD Biosciences, San Jose, CA, USA) was incubated at 10 or 5 μg/ml in DMEM/10% FCS for 30 min and removed by extensive washing before the addition of the parasites in DMEM-BSA.

Techniques: Infection, Activation Assay

Journal: European journal of immunology

Article Title: Expression of the class A macrophage scavenger receptor on specific subpopulations of murine dendritic cells limits their endotoxin response.

doi: 10.1002/eji.200535660

Figure Lengend Snippet: Figure 7. Unaltered expression of CD14 and TLR4-MD-2 on SR- A–/– BM-DC. Two-colour FACS profiles of 129/ICR or SR-A–/–

Article Snippet: BM-DC or CD11c+ splenic cells were harvested and washed with FACS buffer (PBS containing 10 mM sodium azide and 0.1% BSA) and incubated for 30 min at 4 C with the appropriate dilution of primary antibody for SR-A (2F8, Serotec), MHC II (M5/114.1, BD Biosciences), CD11b (Ly-40, Serotec), CD80 (B7-1/16-10A1, BD Biosciences), CD86 (B7-2/ 6L1, BD Biosciences), CD8a (Ly-2/53-6.7, BD Biosciences), CD11c (HL3, BD Biosciences), CD4 (YTS191.1, Serotec), CD103 (M290, BD Biosciences), B220 (RA3-6B2, BD Biosciences), DEC-205 (NLDC-145, Serotec), CD40 (3/23, Serotec), TNF-a (MP6-XT22, BD Biosciences), TLR4-MD-2 (MTS510, Serotec) or isotype matched controls (all from Serotec).

Techniques: Expressing