The leader region of Laminin B1 mRNA confers cap-independent translation

Translation initiation of eukaryotic mRNAs generally occurs by cap-dependent ribosome scanning. However, certain mRNAs contain internal ribosome entry sites (IRES) allowing cap-independent translation. Several of these IRES-competent transcripts and their corresponding proteins are involved in tumourigenesis. This study focused on IRES-driven translation control during the epithelial to mesenchymal transition (EMT) of hepatocytes that reflects crucial aspects of carcinoma progression. Expression profiling of EMT revealed Laminin B1 (LamB1) to be translationally upregulated. The 5′-untranslated region (UTR) of LamB1 was potent to direct IRES-dependent mRNA utilization of a bicistronic reporter construct. Stringent assays for cryptic promoter and splice sites showed no aberrantly expressed transcripts, suggesting that the reporter activity provided by the leader region of LamB1 mRNA exclusively depends on IRES. In accordance, LamB1 expression increased upon negative interference with cap-dependent translation by expression of human rhinovirus 2A protease or heat shock of cells. Finally, the enhanced expression of LamB1 during EMT correlated with an elevated IRES activity. Together, these data provide first evidence that the 5′-UTR of LamB1 contains a bona fide IRES that directs translational upregulation of LamB1 during stress conditions and neoplastic progression of hepatocytes

Canakinumab for Treatment of Adult-Onset Still's Disease to Achieve Reduction of Arthritic Manifestation (CONSIDER): phase II, randomised, double-blind, placebo-controlled, multicentre, investigator-initiated trial

Background: Inhibition of interleukin (IL)-1 represents a promising treatment option in adult-onset Still's disease (AOSD). Objective: To investigate the efficacy and safety of canakinumab in patients with AOSD and active joint involvement by means of a multicentre, double-blind, randomised, placebo-controlled trial. Methods Patients with AOSD and active joint involvement (tender and swollen joint counts of >= 4 each) were treated with canakinumab (4 mg/kg, maximum 300 mg subcutaneous every 4 weeks) or placebo. The primary endpoint was the proportion of patients with a clinically relevant reduction in disease activity at week 12 as determined by the change in disease activity score (Delta DAS28>1.2). Results At enrolment, patients had high active disease with a mean DAS28(ESR) of 5.4 in the canakinumab and 5.3 in the placebo group, respectively. In the intention-to-treat analysis, 12 patients (67%) in the canakinumab group and 7 patients (41%) in the placebo group fulfilled the primary outcome criterion (p=0.18). In the per-protocol analysis, significantly higher American College of Rheumatology (ACR) 30% (61% vs 20%, p=0.033), ACR 50% (50% vs 6.7%, p=0.009) and ACR 70% (28% vs 0%, p=0.049) response rates were observed in the canakinumab group compared with the placebo group. Two patients in the canakinumab group experienced a serious adverse event. Conclusion Although the study was terminated prematurely and the primary endpoint was not achieved, treatment with canakinumab led to an improvement of several outcome measures in AOSD. The overall safety findings were consistent with the known profile of canakinumab. Thus, our data support indication for IL-1 inhibition with canakinumab in AOSD

LamB1 translation after heat shock or intervention with ribosome scanning through 2A protease-dependent cleavage of eIF4G

<p><b>Copyright information:</b></p><p>Taken from "The leader region of Laminin B1 mRNA confers cap-independent translation"</p><p></p><p>Nucleic Acids Research 2007;35(8):2473-2482.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1885646.</p><p>© 2007 The Author(s)</p> () LamB1 expression in MIM-R cells 4, 6 and 8 h after heat shock as detected by western blotting. () Firefly luciferase assay of MIM-R hepatocytes transfected either with pR-EMCV-F or pR-Lam-F bicistronic plasmids. Cells were exposed to heat shock 12 h post-transfection. 48 hours after transfection, Firefly luciferase activity was determined and normalized to the RNA level after reverse transcription and quantitation of cDNA. () Western blot analysis of MIM-R cells showing the cleavage of eIF4GI/II. MIM-R cells were transfected with wild-type 2A protease expressing plasmid (p2Awt) and lysed at the indicated times. () Firefly luciferase assay of MIM-R hepatocytes co-transfected with pR-F and either p2Amut or p2Awt. () Firefly luciferase assay of MIM-R cells co-transfected either with pR-EMCV-F or pR-Lam-F and p2Amut or p2Awt, respectively. Cells were lysed 48 h post-transfection and the Firefly luciferase activity was normalized to the RNA level after reverse transcription and quantitation of cDNA

Cell signaling pathways activated by oncolytic influenza A viruses in melanoma cells.

<p><b>A</b>) Colo-679 cells were infected with IVR-116, delNS1 or delNS1-IL-15 respectively (MOI 1). Protein levels of ERK1/2, phosphorylated ERK1/2 (pERK1/2), AKT, phosphorylated AKT (pAKT) and β-Actin were determined 24 hpi by Western blot. <b>B–D</b>) The role of ERK1/2 phosphorylation in melanoma cell oncolysis by delNS1. Colo-679 cells were transfected with siRNA directed against ERK1, ERK2, or non-target siRNA and infected at MOI 1 with delNS1 48 hpi. <b>B</b>) Western blot analysis of ERK1 and ERK2 levels. Western blot extracts were taken 24 hpi. <b>C</b>) The cell viability was assesed 48 hpi by MTT assay. <b>D</b>) Virus titres were determined as TCID₅₀/mL 24 hpi.</p

Apoptosis induction in influenza A virus-infected melanoma cells.

<p>Cells were infected at MOI 1. 10 µg/mL Cisplatin (CDDP) were used as a control for apoptosis induction. <b>A</b>) The percentage of cells in sub-G1-phase was determined 48 hpi. <b>B–E</b>) Caspase 3/7, 8, or 9 activity was determined 24 hpi. *significantly different compared to mock, P<0.01, **significantly different compared to delNS1 infection, P<0.01.</p

Expression of biological active interleukin-15.

<p><b>A</b>) The cell lines were infected with delNS1 or delNS1-IL-15 at MOI 1 or non-infected (mock). Supernatants were analysed for interleukin-15 by ELISA after the indicated incubation periods. <b>B</b>) Induction of natural killer (NK) cell mediated tumour cell lysis. Primary human NK-cells were incubated with cell culture supernatants of mock, delNS1-, or delNS1-IL-15-infected Colo-679 cells. After four days of incubation, NK-cells were analysed for cytotoxicity against non-infected tumour target cells. NK-cells incubated without or with 100 U recombinant IL-15 served as control.</p

Infection rate and replication kinetics of influenza A viruses.

<p><b>A</b>) Immune staining of infected cells. Cells were infected at MOI 1, 0.1 or 0.01 respectively. Staining for influenza A virus NP antigen expression was performed 24 hours post infection. <b>B</b>) Cells were infected with IVR-116, delNS1, or delNS1-IL-15 (MOI 0.1). At the given time points, aliquots of the supernatants were taken and the TCID₅₀/mL was determined.</p