Total Hsp27 (A), phospho-Hsp27 (B) and IL-8 (C) were measured in serum samples of healthy volunteers, patients with CRC lung metastases before and 3 months after metastasectomy (each n = 10; whiskers indicate standard deviation).

<p>Total Hsp27 (A), phospho-Hsp27 (B) and IL-8 (C) were measured in serum samples of healthy volunteers, patients with CRC lung metastases before and 3 months after metastasectomy (each n = 10; whiskers indicate standard deviation).</p

Representative images showing pulmonary metastases with low, intermediate and high intensity of positive tumor stroma stained for Hsp27 and α-SMA.

<p>Stromal fibroblasts were further identified by vimentin staining. (DAB substrate, same tumor specimen per row, 200x magnification).</p

The degree of Hsp27 expression score in the tumor stroma correlated significantly with the expression of stromal α-SMA (A).

<p>CD31-positive microvessels surrounded by tumor stroma next to tumor cells (asterisk) (B). MVD was significantly increased in primary tumors and metastases with strong stromal Hsp27 expression (C). Immunofluorescence showed a co-expression of stromal Hsp27 and α-SMA especially in PM (400x magnification) (D).</p

Kaplan-Meier plots showing the lung-metastasis free survival, recurrence free survival and overall survival after metastasectomy dependent on stromal Hsp27 (A), stromal α-SMA (B) and tumor Hsp27 (C) scoring.

<p>Kaplan-Meier plots showing the lung-metastasis free survival, recurrence free survival and overall survival after metastasectomy dependent on stromal Hsp27 (A), stromal α-SMA (B) and tumor Hsp27 (C) scoring.</p

Beneficial long term results of ATG treatment six weeks after AMI.

<p>(a) Hearts obtained from untreated control animals evidenced an extensive scar size and signs of ventricular dilation, whereas scar size was reduced in ATG injected rats with a better preserved ventricular geometry (b). ATG injection after AMI resulted in a reduction of scar size from 24.95% of the left ventricle in untreated controls to 11.43% in the treatment group, p<0.01, n = 9–13 per group). Parameters of cardiac function (ejection fraction, shortening fraction, left ventricular end-diastolic diameter and left ventricular end-systolic diameter) were significantly improved by ATG (d,e,f,g).</p

Beneficial short term effects of ATG in a rat AMI model three days after ligation of the left anterior descending artery.

<p>Compared to untreated controls (a) H&E stained specimens of infarcted myocardium from ATG treated rats showed much denser cellular infiltrates (b). The total infarcted area three days after induction of AMI was significantly reduced by ATG (c). Immuhistology showed an increased homing of CD68 positive monocytes/macrophages after ATG treatment (e,f) compared to controls (d).</p

Induction of cytokine secretion by ATG in cell cultures of human PBMC.

<p>ATG showed a significant induction of pro- and anti-inflammatory cytokine secretion. Moreover, ATG also triggered the release of pro-angiogenic factors such as VEGF and CXC and CC chemokines (e.g. IL-8, GRO-alpha, ENA-78, MCP-1) in a time dependent fashion.</p>*<p>p<0.05 vs. matched controls.</p>†<p>p<0.01 vs. matched controls.</p>‡<p>p<0.001 vs. matched controls.</p

Accumulation of cytokines, chemokines and growth factors in whole blood.

<p>ELISA analysis showed that ATG induced a significant dose dependent increase in the secretion of pro- and anti-inflammatory cytokines compared to untreated PBMC after 24 hours of culture. To an even greater extent the release of pro-angiogenic chemokines (e.g. IL-8, GRO-alpha, ENA-78, MCP-1) was triggered. The use of an IVIG preparation failed to initiate the secretion of the aforementioned factors.</p>*<p>p<0.05 vs. 24 h.</p>†<p>p<0.01 vs. 24 h.</p>‡<p>p<0.001 vs. 24 h.</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 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