Blocking experiments with anti-β7, anti-α4 and anti-αVβ3 antibodies for adhesion to HS-5 stromal cells.

<p>Panel A, Blocking with anti-β7. HMCLs were stimulated with Pam3CSK4 for 24 hours and then treated with anti-β7 antibody before adhesion to HS5-coated wells. Panel B, Blocking experiments with anti-α4 and anti-αVβ3 antibodies for adhesion to HS-5. HMCLs were stimulated with Pam3CSK4 for 24 hours and then treated with anti-α4 and anti-αVβ3 antibodies before adhesion to HS5-coated wells. The results are the statistical analyses of data in 3 separate experiments expressed as mean ± SEM, *<i>P<0.05, **P<0.01, ***P<0.001.</i></p

The effect of Pam3CSK4 on adhesion of HMCLs to BMSCs.

<p>HMCLs were stimulated with Pam3CSK4 for 24 hours and then exposed to HS5-coated wells for adhesion assay as explained in materials and methods. Pam3CSK4 decreased adhesion of OPM-1, OPM-2, and NCI-H929 cell lines to HS-5 in a dose-dependent manner. On the contrary, adhesion of L363, UM-6, UM-9 and U266 cell lines increased dose-dependently. The results are the statistical analyses of data in at least 3 separate experiments expressed as mean ± standard error of mean, *<i>P<0.05, **P<0.01, ***P<0.001.</i></p

Pam3CSK4 induces apoptosis enhancing effects in bortezomib-treated HMCLs partly through caspase-3 activation.

<p>L363 (panel <b>A</b>), OPM-2 (panel <b>B</b>) and U266 (panel <b>C</b>) were stimulated for 24 hours with Pam3CSK4, adhered to primary human bone marrow isolated stromal cells and then exposed to drug treatment and cleaved caspase-3 FACS analysis, as explained in materials and methods. Pam3CSK4 alone weakly induced cleaved caspase-3 expression (compared to baseline) and its combination with bortezomib induced a higher level of cleaved caspase-3 protein in L363 and OPM-2 cell lines. Data are representative for the analysis of two MM primary BMSC samples.</p

TLR-1/2 activation effects on expression of β7, αVβ3 and α4 integrins in HMCLs.

<p>HMCLs were stimulated with Pam3CSK4 for 24 hours and then applied to FACS analysis for expression of integrin molecules as explained in materials and methods. Pam3CSK4 downregulated β7 expression in Fravel, OPM-1, OPM-2, and NCI-H929 in a dose-dependent manner. No change in β7 expression was observed in any other cell line. Pam3CSK4 up-regulated αVβ3 and α4 expression dose-dependently in all HMCLs except RPMI-8226. The results are the statistical analyses of data in 3 separate experiments expressed as mean ± SEM, *<i>P<0.05, **P<0.01, ***P<0.001</i>. (Ust: Unstimulated).</p

The apoptosis enhancing effect of Pam3CSK4 on HMCLs in the context of HS-5 stromal cells.

<p>Apoptosis in L363 (panel <b>A</b>), OPM-2 (panel <b>B</b>) and U266 (panel <b>C</b>) was determined by flow cytometric analysis of annexin-V binding. Percentage of apoptotic cells was calculated by selecting the gated CD138 positive cells. Left panels are one representative out of three separate experiments for each cell line and the right panels are statistical analyses of all experiments. HMCLs were stimulated for 24 hours with Pam3CSK4, adhered to HS-5 cells and then exposed to drug treatment as explained in materials and methods. The results are the statistical analyses of data in 3 separate experiments expressed as mean ± SEM, *<i>P<0.05, **P<0.01, ***P<0.001.</i></p

data_sheet_2_Predictable Irreversible Switching Between Acute and Chronic Inflammation.zip

<p>Many a disease associates with inflammation. Upon binding of antigen-antibody complexes to immunoglobulin-like receptors, mast cells release tumor necrosis factor-α and proteases, causing fibroblasts to release endogenous antigens that may be cross reactive with exogenous antigens. We made a predictive dynamic map of the corresponding extracellular network. In silico, this map cleared bacterial infections, via acute inflammation, but could also cause chronic inflammation. In the calculations, limited inflammation flipped to strong inflammation when cross-reacting antigen exceeded an “On threshold.” Subsequent reduction of the antigen load to below this “On threshold” did not remove the strong inflammation phenotype unless the antigen load dropped below a much lower and subtler “Off” threshold. In between both thresholds, the network appeared caught either in a “low” or a “high” inflammatory state. This was not simply a matter of bi-stability, however, the transition to the “high” state was temporarily revertible but ultimately irreversible: removing antigen after high exposure reduced the inflammatory phenotype back to “low” levels but if then the antigen dosage was increased only a little, the high inflammation state was already re-attained. This property may explain why the high inflammation state is indeed “chronic,” whereas only the naive low-inflammation state is “acute.” The model demonstrates that therapies of chronic inflammation such as with anti-IgLC should require fibroblast implantation (or corresponding stem cell activation) for permanence in order to redress the irreversible transition.</p

data_sheet_1_Predictable Irreversible Switching Between Acute and Chronic Inflammation.pdf

<p>Many a disease associates with inflammation. Upon binding of antigen-antibody complexes to immunoglobulin-like receptors, mast cells release tumor necrosis factor-α and proteases, causing fibroblasts to release endogenous antigens that may be cross reactive with exogenous antigens. We made a predictive dynamic map of the corresponding extracellular network. In silico, this map cleared bacterial infections, via acute inflammation, but could also cause chronic inflammation. In the calculations, limited inflammation flipped to strong inflammation when cross-reacting antigen exceeded an “On threshold.” Subsequent reduction of the antigen load to below this “On threshold” did not remove the strong inflammation phenotype unless the antigen load dropped below a much lower and subtler “Off” threshold. In between both thresholds, the network appeared caught either in a “low” or a “high” inflammatory state. This was not simply a matter of bi-stability, however, the transition to the “high” state was temporarily revertible but ultimately irreversible: removing antigen after high exposure reduced the inflammatory phenotype back to “low” levels but if then the antigen dosage was increased only a little, the high inflammation state was already re-attained. This property may explain why the high inflammation state is indeed “chronic,” whereas only the naive low-inflammation state is “acute.” The model demonstrates that therapies of chronic inflammation such as with anti-IgLC should require fibroblast implantation (or corresponding stem cell activation) for permanence in order to redress the irreversible transition.</p

Crosslinking is required for elicitation of allergic ear swelling responses.

<p>Mice were intradermal injected in the right ears with either 500 ng TNP-Ig-fLC (A and B) or 500 ng TNP-specific IgE (C and D). At 20 hours after sensitization, mice were intravenously injected with either 100 µg DNP-HSA (panel A and C) or 100 µg DNP-HSA combined with 750 µg DNP-Ala (50× molar excess monovalent antigen) (panel B and D). Ear swelling was measured at 10, 30 and 60 minutes after antigen injection and compared to ear thickness before antigen challenge. DNP-HSA administration caused a significant increase in ear swelling in both Ig-fLC and IgE sensitized mice (panel A and C). Challenge with DNP-HSA combined with DNP-L-Ala prevented development of ear swelling for both Ig-fLC- (panel A vs B) and IgE-mediated (panel C vs D) responses. All treatment groups consisted of 5 mice. Values are mean±SE.</p

Real-time analysis of TNP-specific Ig-fLC binding to TNP-BSA coated SPR chips.

<p>Increasing concentrations of Ig-fLC (300 ng, 900 ng, 1.8 µg, 2.7 µg and 3.6 µg per CM5 sensor chip) resulted in increased binding curves (A). Administration of TNP-IgE (3.6 µg) resulted in significant binding to the TNP-coated surface and saturation around 350 s. Administration of equal amounts of TNP-Ig-fLC showed binding, yet no clear saturation was observed (B). Administration of TNP-BSA (1 µg/ml) completely eliminated the binding of Ig-fLC to the antigen-coated chip (C). No loss in binding was observed after non-conjugated BSA administration (data not shown). Results are representative for 3 individual experiments.</p

Binding of Alexa633-labeled DNP-HSA to TNP-specific Ig-fLCs bound to mast cells.

<p>Murine bone marrow-derived mast cells (BMMC) were incubated with TNP-specific Ig-fLC (6 µg) or IgE (1 µg). After incubation, Alexa633-labeled DNP-HSA (300 ng) was added. Administration of Alexa633-labeled DNP-HSA resulted in increased binding to BMMCs sensitized with TNP-specific Ig-fLC (B, middle) or IgE (B, right) as compared to non-sensitized BMMC (B, left panel). To evaluate whether the binding of A633-DNP-HSA was specific, a 30-fold excess of unlabeled DNP-HSA (10 µg) was administrated simultaneously with the Alexa633 labeled-DNP-HSA. This resulted in a detectable loss in binding to Ig-fLC- and IgE-sensitized BMMCs (C). Panel A depicts untreated BMMC. Results are representative for 3 independent experiments.</p