Th17 cells in multiple sclerosis express higher levels of JAK2, which increases their surface expression of IFN-γR2.

IFN-ß inhibits the expansion of Th17 cells in active multiple sclerosis (AMS), and this might contribute to improve the clinical symptoms. The effectiveness of this inhibition, however, requires intact IFN-γ signaling in T cells. In this study, we report that both mRNA and cell surface expression of the signaling chain of the IFN-γ receptor (IFN-γR2) and its cognate tyrosine kinase JAK2 are enhanced in peripheral blood Th17 cells and clones from patients with AMS compared with those with inactive multiple sclerosis (IMS) or healthy subjects (HS). IFN-γ decreased the frequency of Th17 peripheral cells and proliferation of Th17 clones from AMS patients. Stimulation of PBMCs from HS in Th17-polarizing conditions resulted in the enhancement of JAK2 expression and accumulation of cell surface IFN-γR2. The role of JAK2 in the modulation of IFN-γR2 was demonstrated as its transduction prevented rapid internalization and degradation of IFN-γR2 in JAK2-deficient γ2A cells. In conclusion, these data identify JAK2 as a critical factor that stabilizes IFN-γR2 surface expression in Th17 cells from AMS patients, making them sensitive to IFN-γ. These data may have clinical implications for a better use of IFNs in multiple sclerosis and possibly other inflammatory diseases.

Synergistic anti-proliferative and pro-apoptotic activity of combined therapy with bortezomib, a proteasome inhibitor, with anti-epidermal growth factor receptor (EGFR) drugs in human cancer cells.

The proteasome plays a pivotal role in the turnover of regulatory transduction proteins induced by activated cell membrane growth factor receptors. The epidermal growth factor receptor (EGFR) pathway is crucial in the development and progression of human epithelial cancers. Proteasome inhibition may sensitize human cancer cell lines to EGFR inhibitors. We investigated the growth inhibitory and pro-apoptotic effects of the proteasome inhibitor bortezomib in combination with anti-EGFR drugs, such as gefitinib, vandetanib, and cetuximab in EGFR-expressing human cancer cell lines. Bortezomib determined dose-dependent growth inhibition in a nine cancer cell line panel (IC(50) values, range 6-42 nM). A significant synergistic growth inhibitory effect was observed with the combination of bortezomib and each EGFR inhibitor in all cell lines (combination index, CI, range 0.10-0.55), which was accompanied by a significant induction in apoptosis by the combined treatment with bortezomib, cetuximab and vandetanib. In HCT-116 colon cancer and A549 lung adenocarcinoma cells, bortezomib plus EGFR inhibitor treatment induced a more effective inhibition of EGFR-activated down-stream signals, including a marked suppression in activated, phosphorylated Akt (P-Akt). In contrast, overexpression of a constitutively active P-Akt protected A549 cells by cell growth inhibition and apoptosis following treatment with bortezomib and EGFR inhibitors. The combined treatment with bortezomib and EGFR inhibitors has a synergistic growth inhibitory and pro-apoptotic activity in different human cancer cells which possess a functional EGFR-dependent autocrine growth pathway through to a more efficient and sustained inhibition of Akt.

Myocardial strain analysis in a doxorubicin-induced cardiomyopathy model.

The aim of our study was to determine if strain (S) and strain rate (SR) imaging are more sensitive indices with respect to standard echocardiographic parameters to assess cardiac function in an experimental model of doxorubicin (DOX)-induced cardiomyopathy. DOX was administered intraperitoneally 4x/wk at the dose of 1.25 mg/kg/d over four weeks in Wistar rats (n = 26). Other 14 Wistar rats were used as controls. Echocardiographic studies were performed before DOX treatment (baseline), after two and four weeks of treatment. At two and four weeks of DOX treatment, rat hearts were collected for histologic analysis. After two weeks of DOX treatment, there were no significant changes in standard echocardiographic parameters and in myocardial velocities, but after four weeks of treatment, ejection fraction significantly decreased and left ventricle dimensions significantly increased. Also, myocardial velocities were significantly reduced after four weeks of treatment. Conversely, S and SR values changed significantly already after two weeks of treatment. At baseline, S and SR values were 27 +/- 7% and 7.4 +/- 1.7, respectively, and they significantly decreased to 12 +/- 6% (p < 0.0001) and 6 +/- 1.5 (p < 0.005) at two weeks of treatment. These changes were also significant compared with control rat parameters (p < 0.001). At four weeks of DOX treatment, a progressive worsening occurred. Strain and SR further significantly decreased to 10 +/- 4% (p < 0.0001 vs. baseline) and 4.6 +/- 1.6 (p < 0.0001 vs. baseline), respectively. These changes were supported by histologic findings. Indeed, damage in myocardial tissue was demonstrated by histology already after two weeks of DOX treatment, with a damage progression after four weeks of treatment. Our data demonstrate that S and SR imaging are more sensitive indices in identifying early myocardial systolic changes induced by DOX than standard echocardiographic parameters and myocardial velocities. We believe that our model will be very supportive to further assess these new diagnostic strategies to provide a precocious diagnosis of LV dysfunction with a unique opportunity to initiate preventive cardioactive therapy. (E-mail: giodisal@yahoo.it).

Synergistic antitumor activity of sorafenib in combination with epidermal growth factor receptor inhibitors in colorectal and lung cancer cells.

PURPOSE: Cancer cell survival, invasion, and metastasis depend on cancer cell proliferation and on tumor-induced angiogenesis. We evaluated the efficacy of the combination of sorafenib and erlotinib or cetuximab. EXPERIMENTAL DESIGN: Sorafenib, erlotinib, and cetuximab, alone or in combination, were tested in vitro in a panel of non-small cell lung cancer (NSCLC) and colorectal cancer cell lines and in vivo in H1299 tumor xenografts. RESULTS: Epidermal growth factor receptor (EGFR) ligand mRNAs were expressed in all NSCLC and colorectal cancer cell lines with variable levels ranging from 0.4- to 8.1-fold as compared with GEO colorectal cancer cells. Lung cancer cells had the highest levels of vascular endothelial growth factors (VEGF) A, B, and C, and of VEGF receptors as compared with colorectal cancer cells. Combined treatments of sorafenib with erlotinib or cetuximab produced combination index values between 0.02 and 0.5, suggesting a significant synergistic activity to inhibit soft agar colony formation in all cancer cell lines, which was accompanied by a marked blockade in mitogen-activated protein kinase and AKT signals. The in vitro migration of H1299 cells, which expressed high levels of both VEGF ligands and receptors, was inhibited by treatment with sorafenib, and this effect was significantly increased by the combination with anti-EGFR drugs. In nude mice bearing established human H1299 xenografts, treatment with the combination of sorafenib and erlotinib or cetuximab caused a significant tumor growth delay resulting in 70 to 90 days increase in mice median overall survival as compared with single-agent sorafenib treatment. CONCLUSIONS: Combination treatment with sorafenib and erlotinib or cetuximab has synergistic antitumor effects in human colorectal and lung cancer cells.

beta(3) Integrin subunit mediates the bone-resorbing function exerted by cultured myeloma plasma cells.

alpha(v)beta(3) integrin was investigated in multiple myeloma in relation to the in vitro osteoclast-like activity of malignant plasma cells. Myeloma cells from patients with skeleton involvement overexpressed alpha(v)beta(3) and produced erosion pits on bone substrates, whereas this effect was not observed by cells from patients with no evidence of bone disease. We therefore explored the alpha(v)beta(3) transcriptional pathway in the bone-resorbing cells. Silencing of beta(3) chain abrogated the ability to produce erosion pits and extracellular signal-regulated kinase 1/2 phosphorylation resulting in the defective function of cFos and nuclear factor activator T cell 1, the terminal effectors of osteoclast activation. A similar defect occurred in constitutively beta(3)-deficient cells from patients with no skeleton disease. Microarray gene analysis of beta(3)(+) myeloma cells showed that several osteoclast-related genes were up-regulated. Their functions include the activation of receptor pathways beta(3) and c-fms that regulate several osteoclast functions. These data emphasize the postulated role of myeloma cells in multiple myeloma bone disease and suggest that their osteoclast-like activity is regulated, at least in vitro, by the beta(3) subunit of the integrin.

Human interleukin 17-producing cells originate from a CD161+CD4+ T cell precursor.

We demonstrate that CD161 is a highly up-regulated gene in human interleukin (IL) 17 T helper cell (Th17) clones and that all IL-17-producing cells are contained in the CD161(+) fraction of CD4(+) T cells present in the circulation or in inflamed tissues, although they are not CD1-restricted natural killer T cells. More importantly, we show that all IL-17-producing cells originate from CD161(+) naive CD4(+) T cells of umbilical cord blood, as well as of the postnatal thymus, in response to the combined activity of IL-1 beta and IL-23. These findings implicate CD161 as a novel surface marker for human Th17 cells and demonstrate the exclusive origin of these cells from a CD161(+)CD4(+) T cell progenitor.