Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Role of whole saliva in the efficacy of sublingual immunotherapy in seasonal allergic rhinitis.

doi: 10.1016/j.alit.2018.07.008

Figure Lengend Snippet: Fig. 4. Role of TLR2 and TLR4 signaling in whole saliva-induced IL-10 production by monocytes. (A) Effect of Pam3CSK4, Poly (I:C), LPS, and CpG on IL-10 production. (BeD) Effect of TL2.1 alone (B), PMB alone (C), and TL2.1 plus PMB (D) on IL-10 production. Bar represents the median. P-values were determined using the Dunn test (A; **P < 0.01) or the Wilcoxon signed-rank test (BeD).

Article Snippet: We purchased the following materials for the study: polymyxin B sulfate salt (PMB, SigmaeAldrich, St. Louis, MO), Pam3CSK4, Poly(I:C), LPS, CpG and mouse anti-human TLR2 mAb (TL2.1, Novus Biologicals, Littleton, CO), mouse IgG2a (R&D Systems, Minneapolis, MN); FITC-conjugated anti-human CD80 mAb (BD Biosciences, San Jose, CA), FITC-conjugated anti-human CD86 mAb (Agilent Technologies, Santa Clara, CA), PE-conjugated anti-human HLA-DR mAb (BD Biosciences), and FicollePaque (GE Healthcare BioSciences AB, Uppsala, Sweden).

Techniques:

Journal:

Article Title: Pattern of Cytokine Responses to Gram-Positive and Gram-Negative Commensal Bacteria Is Profoundly Changed when Monocytes Differentiate into Dendritic Cells

doi: 10.1128/IAI.72.5.2671-2678.2004

Figure Lengend Snippet: Expression of CD14, TLR2, and TLR4 on monocytes (A) and DCs and in the absence (B) or in the presence (E) of 10 ng of IFN-γ per ml after stimulation for 48 h with L. plantarum or E. coli. The percentages of CD14-positive cells and the geometric mean fluorescence intensity for TLR expression, respectively, are indicated in the upper right corners of the panels. The lines show the data for the isotype control antibodies, and the solid histograms show the expression of TLRs. The results shown are the results of one representative experiment of three experiments performed. (C) Effect on V. pavula-induced TNF production from DCs after blocking of CD14, TLR2, or TLR4. DCs (5 × 105 cells per ml) were incubated with 10 μg of blocking antibody per ml or 10 μg of isotype control antibody per ml for 1 h prior to stimulation with bacteria (5 × 106 cells per ml) for 7 h. The results after blocking were expressed as the percentage of TNF production relative to control production for each individual. The bars indicate the mean percentage of positive cells from three donors, and the error bars indicate the standard error of the mean. (D) Positive control for the effectiveness and specificity of the blocking antibodies used. The experiments were performed as described above, and the cells were stimulated with 0.1 μg of LPS per ml or 1 μg of peptidoglycan per ml. a-CD14, anti-CD14; a-TLR2, anti-TLR2; a-TLR4, anti-TLR4.

Article Snippet: For blocking experiments, DCs (5 × 10 5 cells per ml) from three individuals were preincubated with anti-CD14 (UCHM-1; Serotec, Oxford, United Kingdom), anti-TLR2 (TL2.1; Alexis Biochemicals, San Diego, Calif.), anti-TLR4 (HTA125; Serotec), or isotype control antibody (10 μg per ml) for 1 h at 4°C, and this was followed by stimulation with V. parvula (5 × 10 6 cells per ml) for 7 h. Supernatants were collected, and the cells were analyzed for expression of different surface markers by flow cytometry.

Techniques: Expressing, Fluorescence, Blocking Assay, Incubation, Positive Control

Journal: Journal of leukocyte biology

Article Title: Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2.

doi: 10.1189/jlb.0803360

Figure Lengend Snippet: Fig. 7. LTA-mediated effects on PMN depend on CD14 and TLR2. (A) Anti-CD14 prevents the shedding of CD62L. Freshly isolated neutrophils were stained with anti-CD62L-PE (FL2) antibody and analyzed by flow cytometry. Neutrophils were incubated at 5 106/ml for 30 min at RT in medium or with anti-CD14 mAb (10 g/ml) or isotype control. Subsequently, the cells were cultured for 3 h in medium alone or in the presence of 10 g/ml LTA. The dot plots show results from a representative experiment. The percentages of CD62L-negative cells are given for the representative experiment and in brackets, the mean SD of three experiments. (B) Anti-CD14 and anti-TLR2 attenuate the LTA-induced IL-8 release by PMN. Neutrophils (5106/ml) were pretreated with 10 g/ml anti-CD14 mAb, anti-TLR2 mAb, anti-TLR4 mAb, and IgG1 and IgG2a isotype-control mAb or without antibody for 30 min at RT before exposure to 10 g/ml LTA for 24 h. ELISA measured the IL-8 content in the supernatant. Results are mean SD of three experiments. Significant reductions of IL-8 release between treatments with control versus specific antibodies are indicated. (C) Blocking CD14 and TLR2 reduces the antiapoptotic effect of LTA. Neutrophils (5106/ml) were preincubated with anti-CD14 mAb, anti-TLR2 mAb, and anti-TLR4 mAb with IgG1 and IgG2a isotype-control mAb or in the absence of antibody for 30 min at RT before exposure to 10 g/ml LTA for 24 h. The ratio of apoptotic PMN was deter- mined by Annexin-V staining as described in Materials and Methods. The data show the LTA-mediated inhibition of spontaneous PMN apoptosis after the given pretreatments. Results are mean SD of three to four experiments. Significant reductions of apoptosis inhibition between treatments with control versus specific antibodies are indicated.

Article Snippet: Freshly isolated PMN were pretreated with 10 g/ml anti-CD14 mAb [immunoglobulin G (IgG)1; clone 18D11, Diatec, Hamburg, Germany], 10 g/ml anti-TLR2 mAb (IgG2a, clone TL2.1, Imgenex, San Diego, CA), and 10 g/ml anti-TLR4 mAb (IgG2a, clone HAT125, Imgenex) for 30 min at RT and subsequently exposed to LTA.

Techniques: Isolation, Staining, Cytometry, Incubation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Blocking Assay, Inhibition

Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis heat shock protein 60 modulates immune response to PPD by manipulating the surface expression of TLR2 on macrophages.

doi: 10.1111/j.1462-5822.2008.01161.x

Figure Lengend Snippet: Fig. 4. The regulation of IL-12 p40 by Mtbhsp60 is TLR dependent. The PMA-differentiated THP-1 macrophages were either left untreated or pretreated with 10 mg ml-1 of anti-TLR2 mAb or anti-TLR4 mAb or isotype-matched control Ab for 1 h. These cells were then activated with PPD in the presence of 3 mg ml-1 of Mtbhsp60 or GST. Cells were either cultured for 48 h for estimating IL-12 p40 cytokine by EIA (A) or for 1 h to measure c-rel levels in the nuclear extracts by Western blot analysis (B). Results shown are representative of three different experiments.

Article Snippet: In some experiments, PMA-differentiated THP-1 macrophages were pretreated with 10 mg ml-1 anti-human TLR2 mAb (TL2.1, mouse IgG2a) or anti-human TLR4 mAb (HTA125, mouse IgG2a) (both from Imgenex, San Diego, CA) for 1 h at 37°C and then stimulated with PPD (10 mg ml-1) in the presence of 3 mg ml-1 of Mtbhsp60 or GST.

Techniques: Control, Cell Culture, Western Blot

Journal: Cellular microbiology

Article Title: Mycobacterium tuberculosis heat shock protein 60 modulates immune response to PPD by manipulating the surface expression of TLR2 on macrophages.

doi: 10.1111/j.1462-5822.2008.01161.x

Figure Lengend Snippet: Fig. 5. The Mtbhsp60 increases surface expression of TLR2 and exploits the TLR2-triggered signalling to downregulate IL-12 p40 in activated macrophages. The THP-1 macrophages were either treated with PMA (A) or left untreated (B) and then incubated with 3 mg ml-1 Mtbhsp60 for various times. The cells were harvested for measuring the surface expression of TLR2 (upper panel) and TLR4 (lower panel) by FACS. In another experiment, PMA-differentiated THP-1 macrophages were transfected with negative control siRNA or TLR2 siRNA and after 24 h cells were activated with PPD in the presence of 3 mg ml-1 of Mtbhsp60. After 48 h, culture supernatants were harvested to estimate IL-12 p40 induction (C). Results shown are representative of 3–4 different experiments.

Article Snippet: In some experiments, PMA-differentiated THP-1 macrophages were pretreated with 10 mg ml-1 anti-human TLR2 mAb (TL2.1, mouse IgG2a) or anti-human TLR4 mAb (HTA125, mouse IgG2a) (both from Imgenex, San Diego, CA) for 1 h at 37°C and then stimulated with PPD (10 mg ml-1) in the presence of 3 mg ml-1 of Mtbhsp60 or GST.

Techniques: Expressing, Incubation, Transfection, Negative Control