Molecular interaction between bacterial antigens and macrophage receptors studied by atomic force microscopy

Atomic force spectroscopy was used to study interaction strengths between bacterial antigens and receptors on macrophages. This method allowed for a direct comparison of the interaction strengths in different systems studied at the level of single molecules

Effects of ECJ judgments on the principle of res judicata of national bodies of members states

<p><b>Effects of CD36 or SR-A deficiency and of anti-CD14 mAb on TNF-α and RANTES production, stimulated by 1-h incubation on ice with 1</b> μ<b>g/ml S-LPS (A-B) or R-LPS (C-D).</b> (A) Anti-CD14 mAb blocks both TNF-α and RANTES production, stimulated by S-LPS. (B) S-LPS stimulates significantly higher cytokine production in CD36-/- than in WT or SR-A-/- PEMs. (C) R-LPS-stimulated cytokine production is more strongly inhibited by anti-CD14 mAb in CD36-/- than in WT or SR-A-/- PEMs. (D) R-LPS stimulates similar cytokine production in WT, CD36-/- and SR-A-/- PEMs. Graphs show means +SEM from 4–6 independent experiments, each performed in 4 replicates. Data on graphs A and C were analyzed with one-sample t-test, and on graphs B and D with ANOVA, followed by the Dunnett’s test. *, p < 0.05.</p

The role of CD36 as a S-LPS receptor.

<p>(A) Incubation with 5 μg/ml rCD14, but not with rCD36 partially removes bS-LPS from rLBP. (B) rLBP, but not rCD36 or serum depletes rCD14 of bound bS-LPS. (C) rLBP binds very weakly to plate-adsorbed rCD14, but not to rCD36 and this binding is only slightly increased by 1 μg/ml S-LPS. (D) bS-LPS binds much more strongly to plate-adsorbed rCD36 in BSA-PBS than in FCS-PBS. (E) rCD36 binding to plate-adsorbed GA-BSA is inhibited by 0.2 mg/ml LTA, 0.1 mg/ml DS and anti-CD36 mAb, whereas 0.2 mg/ml S-LPS inhibits rCD36 binding only in the absence of serum. Chondroitin sulfate (CS) and control IgA have no effect on the binding. (F) A large portion of bS-LPS bound to rCD36 is removed by serum component(s) distinct from CD14 or LBP. (G) Relative to WT controls, internalization of bS-LPS/pHr-SAV complexes is significantly decreased in CD36-/-, but not SR-A-/- PEMs. Graphs show means ± SEM of 3–5 replicates, obtained in single experiments, each representative of at least 3 similar experiments performed (A-F) or averages + SEM from 4 independent experiments (G). Data were analyzed with the regular (A, B, E and F) or repeated measures (G) ANOVA, followed by the Dunnett’s post-test. *, p < 0.05; OD, optical density.</p

Relative roles played by CD36 and CD14 in LPS loading onto TLR4/MD-2 and in LPS-stimulated cytokine production depend on the LPS chemotype and the presence of serum.

<p>(A) Binding of mAbs to PEMs pre-incubated with the indicated concentrations of S-LPS. (B) Forty-min pre-incubation at 37°C with 200 ng/ml S-LPS, but not with R-LPS in FCS-RPMI more strongly inhibits MTS510 mAb binding to TLR4/MD-2 on CD36-/- than on WT PEMs. In contrast, in BSA-RPMI both S-LPS and R-LPS produce significant inhibition of MTS510 mAb binding only in WT PEMs. (C) Forty-min stimulation in FCS-RPMI with 200 ng/ml S-LPS, but not with R-LPS induces significantly higher TNF-α production in WT than in CD36-/- PEMs (left panel). In contrast, R-LPS, but not S-LPS stimulates significantly lower RANTES production in CD36-/- PEMs (right panel). In BSA-RPMI, CD36-/- PEMs exhibit severe impairment of cytokine production in response to both S-LPS and R-LPS. (D) In both WT and CD36-/- PEMs cytokine production stimulated by 40-min incubation with 200 ng/ml S-LPS in serum-free medium is blocked completely by anti-CD14 mAb. Anti-CD14 mAb also blocks cytokine production stimulated by R-LPS in CD36-/- PEMs, whereas it exerts only partial inhibition in WT PEMs. Data shown on graphs A and D are means +SEM of triplicates, obtained in single experiments, which were performed 2 (A) or 3 (D) times with similar results. Graphs B-C show means +SEM from 4–6 independent experiments, each performed in 4 replicates. *, significant inhibition or difference between WT and CD36-/- PEMs (p < 0.05 in Student’s t-test).</p

S-LPS and R-LPS bind to LBP, CD14 and CD36 with different affinities.

<p>(A) 200 ng/ml bS-LPS binding to plate-adsorbed rCD14 is more effectively inhibited by S-LPS than R-LPS. (B) S-LPS is a more efficient competitor than R-LPS in inhibiting bS-LPS binding to plate-adsorbed rLBP. (C) Lower concentrations of R-LPS than S-LPS are required to inhibit rCD36 binding to plate-adsorbed GA-BSA. (D) rCD36 binds with higher affinity to plate-adsorbed R-LPS than to S-LPS or LTA. (E) Cytokine production stimulated by 5 ng/ml S-LPS in serum-free medium is strongly enhanced by 0.2 μg/ml rLBP and, to a lesser extent, by 2 μg/ml rCD14. (F) Only the combination of rLBP and rCD14 enhances cytokine production in response to 5 ng/ml R-LPS. Graphs show means ± SEM of 3–4 replicates, obtained in single experiments, performed twice with similar results. Data were analyzed with two-tailed and one-sample t-test (B and C) or with ANOVA, followed by the Tukey-Kramer test (E and F); *, p < 0.05.</p