Allergen-specific IgE antibody levels (kUA/L) and CD4 counts in HIV-positive patients with AIDS (#8–11) and in HIV-positive patients with CD4 counts below 200/µl (#12–13).

<p>CD4 counts (right y-axes) and allergen-specific IgE levels (left y-axes) measured in serum samples obtained at different points of time (x-axes) are shown. The usual periods of the grass and tree pollen season is indicated in green and brown, respectively.</p

Demographic, clinical and immunological characterization of ten HIV infected patients with low CD4 counts.

<p>Displayed are age, sex and ranges of CD4 counts for follow-up sera. Allergic symptoms and positive results obtained by skin prick testing and determination of allergen-specific IgE are summarized.</p><p>Abbreviations: F: female; M: male; R: rhinitis; RC: rhinoconjunctivitis; U: urticaria; AB: asthma bronchiale; AP: allergic pharyngitis; SPT: skin prick test; Der p: <i>Dermatophagoides pteronyssinus</i>; n.k.: not known;</p

Allergen-specific IgE antibody levels (kUA/L) and CD4 counts in HIV-positive patients with low CD4 counts (#14–21) for whom sequential serum samples were available.

<p>CD4 counts (right y-axes) and allergen-specific IgE levels (left y-axes) measured in serum samples obtained at different points of time (x-axes) are shown. The usual periods of the grass and tree pollen season is indicated in green and brown, respectively.</p

The PI3-Kinase/mTOR-Targeting Drug NVP-BEZ235 Inhibits Growth and IgE-Dependent Activation of Human Mast Cells and Basophils

<div><p>The phosphoinositide 3-kinase (PI3-kinase) and the mammalian target of rapamycin (mTOR) are two major signaling molecules involved in growth and activation of mast cells (MC) and basophils (BA). We examined the effects of the dual PI3-kinase/mTOR blocker NVP-BEZ235 on growth of normal and neoplastic BA and MC as well as immunoglobulin E (IgE)-dependent cell activation. Growth of MC and BA were determined by measuring ³H-thymidine uptake and apoptosis. Cell activation was determined in histamine release experiments and by measuring upregulation of CD63 and CD203c after challenging with IgE plus anti-IgE or allergen. We found that NVP-BEZ235 exerts profound inhibitory effects on growth of primary and cloned neoplastic MC. In the MC leukemia cell line HMC-1, NVP-BEZ235 showed similar IC₅₀ values in the HMC-1.1 subclone lacking KIT D816V (0.025 µM) and the HMC-1.2 subclone expressing KIT D816V (0.005 µM). Moreover, NVP-BEZ235 was found to exert strong growth-inhibitory effects on neoplastic MC in a xenotransplant-mouse model employing NMR1-Foxn1^nu mice. NVP-BEZ235 also exerted inhibitory effects on cytokine-dependent differentiation of normal BA and MC, but did not induce growth inhibition or apoptosis in mature MC or normal bone marrow cells. Finally, NVP-BEZ235 was found to inhibit IgE-dependent histamine release in BA and MC (IC₅₀ 0.5–1 µM) as well as anti-IgE-induced upregulation of CD203c in BA and IgE-dependent upregulation of CD63 in MC. In summary, NVP-BEZ235 produces growth-inhibitory effects in immature neoplastic MC and inhibits IgE-dependent activation of mature BA and MC. Whether these potentially beneficial drug effects have clinical implications is currently under investigation.</p> </div

Effects of NVP-BEZ235 on proliferation in HMC-1 cells and KU812 cells.

<p>A,B: HMC-1.1 cells (left panels), HMC-1.2 cells (middle panels), and KU812 cells (right panels) were cultured in control medium (Co), control medium with DMSO 1∶1,000 (DMSO Co), or with increasing concentrations (0.001 µM–1 µM) of NVP-BEZ235 (A) or RAD001 (B) at 37°C for 48 hours. Thereafter, ³H-thymidine uptake was measured. Results show the percentage of ³H-thymidine uptake compared to control (Co) and represent the mean±S.D. of three independent experiments in each cell line. Asterisk (*): p<0.05. C: Bone marrow MNC from six patients with SM were incubated in control medium (Co) or in various concentrations of NVP-BEZ235 (as indicated) at 37°C for 48 hours. Then, ³H-thymidine uptake was measured. Results show the percentage of ³H-thymidine uptake compared to Co and represent the mean±S.D. of six donors. Asterisk (*): p<0.05.</p

Effects of NVP-BEZ235 on IgE-mediated histamine release in human BA.

<p>BA obtained from healthy donors (A) or patients allergic to Bet v 1 (B) were preincubated with control medium (Medium Co), various concentrations of NVP-BEZ235 (as indicated), LY294002 (20 µM), or wortmannin (1 µM) at 37°C for 30 minutes. Then, cells were exposed to anti-IgE (1 µg/ml; healthy controls) or recombinant allergen (0.1 µg/ml of Bet v 1; allergic patients) at 37°C for 30 minutes. After centrifugation, histamine concentrations were determined in supernatants and cell-lysates by RIA. Histamine release is expressed as percentage of total histamine. Results represent the mean±S.D. from five donors. Asterisk (*) indicates p<0.05. C: BA obtained from one healthy donor were incubated in control medium (♦-♦) or medium containing NVP-BEZ235, 10 µM (▪-▪) for 30 minutes at 37°C. Then, cells were incubated with various concentrations (as indicated) of anti-IgE (left panel), Ca-ionophore A23187 (middle panel), or recombinant C5a (right panel) for another 30 minutes (37°C). After incubation, supernatants and lysates were harvested and examined for their histamine content. Histamine release was calculated as percent of total histamine. Data are expressed as mean±S.D. of triplicates.</p

Effects of NVP-BEZ235 on mediator secretion in human MC.

<p>A: IgE-preloaded cultured cord blood-derived MC (left panel) and IgE-preloaded lung MC (right panel) were incubated in control medium, various concentrations of NVP-BEZ235 (as indicated), LY294002 (20 µM), or wortmannin (1 µM) (30 minutes, 37°C). Then, cells were challenged with anti-IgE (10 µg/ml) at 37°C for 30 minutes. After incubation, histamine release was determined. Results show the percentage of released histamine and represent the mean±S.D. of four (cultured MC, left panel) or five independent experiments (5 lung MC samples, right panel). Asterisk (*): p<0.05. B: Cultured MC were incubated in the presence of control medium (indicated by black bars) or medium containing NVP-BEZ235, 10 µM (open bars) for 30 minutes at 37°C. Then, cells were incubated with various concentrations of anti-IgE as indicated. After incubation, supernatants and lysates were harvested and examined for histamine content. Histamine release was calculated as percent of total histamine. Results show the percent of released histamine and are expressed as mean±S.D. of three independent experiments. Asterisk (*): p<0.05. C: MC preloaded with NIP(5)-BSA-specific IgE were cultured in control medium (Co) or in various concentrations of NVP-BEZ235 (0.001–10 µM) for 30 minutes at 37°C. Thereafter, cells were washed and incubated with NIP(5)-BSA (100 ng/ml) for 90 minutes (37°C). Then, cell-free supernatants were collected and β-hexosaminidase release was measured. Results are expressed in percent of response induced by anti-IgE without NVP-BEZ235 and represent the mean±S.D of three independent experiments. Asterisk (*) indicates p<0.05.</p

Effects of NVP-BEZ235 on survival of HMC-1 cells and KU812 cells.

<p>A,B: HMC-1.1 cells (left panels), HMC-1.2 cells (middle panels), and KU812 cells (right panels) were cultured in control medium (Co), LY294002 (20 µM), or increasing concentrations (0.001 µM–10 µM) of NVP-BEZ235 (A) or RAD001 (B) at 37°C for 48 hours. Then, the numbers (percentage) of apoptotic cells was counted on Giemsa-stained cytospin slides. Results represent the mean±S.D. of three independent experiments. Asterisk (*) indicates p<0.05. C: HMC-1.1 cells (upper panels), HMC-1.2 cells (middle panels) and KU812 cells (lower panels) were incubated in control medium (Co) (left panels) or in medium containing NVP-BEZ235, 1 µM (middle panels) or RAD001, 1 µM (right panel) for 48 hours. After incubation, cells were examined for combined AnnexinV/PI staining by flow cytometry. D: HMC-1.1 (left panel), HMC-1.2 (middle panel), and KU812 cells (right panel) were incubated in control medium (Co) or in various concentrations of NVP-BEZ235 or RAD001 as indicated (37°C, 48 hours). Then, the percentage of active caspase 3-positive cells was determined by flow cytometry. Results represent the mean±S.D. of three independent experiments. Asterisk (*) indicates p<0.05. E: HMC-1.1 cells (upper panels), HMC-1.2 cells (middle panels) and KU812 cells (lower panels) were incubated in control medium (Co) or in medium containing NVP-BEZ235 (1 or 10 µM) or RAD001 (1 or 10 µM) (as indicated) at 37°C for 48 hours. Thereafter, cells were harvested and subjected to Tunel assay and examined by fluorescence microscopy.</p

Effects of NVP-BEZ235 on expression of activation-linked cell surface antigens on BA and MC.

<p>A: BA in whole blood samples were preincubated in control medium (Medium Co) or in medium containing various concentrations of NVP-BEZ235 (0.001–10 µM), LY294002 (20 µM), or wortmannin (1 µM) at 37°C for 15 minutes. Then, cells were exposed to anti-IgE antibody E-124.2.8 (1 µg/ml) for another 15 minutes (37°C). Thereafter, cells were stained with mAb directed against CD63 (left panel) or CD203c (right panel), and analyzed by multicolor flow cytometry as described in the text. BA were defined as CD203c-positive cells in all samples. Anti-IgE-induced upregulation of CD antigens was calculated from mean fluorescence intensities (MFIs) obtained with stimulated (MFIstim) and unstimulated (MFIcontrol) cells and was expressed as SI (MFIstim∶MFIcontrol). Results show SI values and represent the mean±S.D. from five normal donors (the same as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029925#pone-0029925-g001" target="_blank">Figure 1A</a>). Asterisk (*) indicates p<0.05. B: IgE-preloaded cultured MC (left panel) or IgE-preloaded lung MC (right panel) were incubated in control medium (Co) or in various concentrations of NVP-BEZ235 (as indicated) at 37°C for 40 minutes. Then, cells were incubated with anti-IgE antibody E-124.2.8 (10 µg/ml) for 30 minutes (37°C). After incubation, expression of CD63 (♦-♦) and CD203c (□-□) on MC was analyzed by flow cytometry. Results show the percent-inhibition of anti-IgE-induced upregulation of CD63 or CD203c on MC, and represent the mean±S.D. from three (cultured MC) or five (lung MC) independent experiments. Asterisk (*): p<0.05. C: Ficoll-isolated BA (left panel) and lung MC (right panel) were preincubated in control medium (Co) or with 10 µM NVP-BEZ235 at 37°C for 15 minutes. Then, cells were exposed to control medium or anti-IgE antibody E-124.2.8 (1 µg/ml) for another 15 minutes (37°C). Thereafter, cells were stained with mAb directed against CD63 or CD203c and analyzed by flow cytometry using a multicolor surface staining protocol and a cytoplasmic staining protocol. Results show SI values of surface CD63 (white bars), cytoplasmic CD63 (black bars), surface CD203c (light grey bars) and cytoplasmic CD203c (dark grey bars) and represent the mean±S.D. from three independent experiments.</p

Effects of NVP-BEZ235 on expression of pAkt, pS6, and pSTAT5 in HMC-1 cells and KU812 cells.

<p>A: HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel) were incubated with control medium (Co) or medium containing NVP-BEZ235 (1 or 10 µM) or RAD001 (1 or 10 µM) at 37°C for 4 hours. Thereafter, cells were permeabilized and stained with antibodies against pAkt (S473), pS6, or pSTAT5 as described in the text. Expression of phosphorylated (p) signaling molecules in HMC-1 cells and KU812 cells were determined by flow cytometry. Results show the staining index (mean fluorescence intensity values corrected for the isotype) and represent the mean±S.D. from three independent experiments. B: HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel) were cultured in the absence or presence of NVP-BEZ235 (1 µM) RAD001 (1 µM) at 37°C for 4 hours. Thereafter, cells were lysed and Western blotting was performed using antibodies against pAkt (S473), Akt, pSTAT5, STAT5, pS6 and β-Actin. C: Western blot results were quantified by densitometry in HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel). Results represent the mean±S.D. from three independent experiments.</p