Estimation of Nuwiq® (simoctocog alfa) activity using one-stage and chromogenic assays-Results from an international comparative field study.

BACKGROUND Accurate determination of coagulation factor VIII activity (FVIII:C) is essential for effective and safe FVIII replacement therapy. FVIII C can be measured by one-stage and chromogenic substrate assays (OSAs and CSAs, respectively); however, there is significant interlaboratory and interassay variability. AIMS This international comparative field study characterized the behaviour of OSAs and CSAs used in routine laboratory practice to measure the activity of Nuwiq® (human-cl rhFVIII, simoctocog alfa), a fourth-generation recombinant human FVIII produced in a human cell line. METHODS FVIII-deficient plasma was spiked with Nuwiq® or Advate® at 1, 5, 30 and 100 international units (IU)/dL. Participating laboratories analysed the samples using their routine procedures and equipment. Accuracy, inter- and intralaboratory variation, CSA:OSA ratio and the impact of different OSA and CSA reagents were assessed. RESULTS Forty-nine laboratories from 9 countries provided results. Mean absolute FVIII:C was comparable for both products at all concentrations with both OSA and CSA, with interproduct ratios (Nuwiq® :Advate® ) of 1.02-1.13. Mean recoveries ranged from 97% to 191% for Nuwiq® , and from 93% to 172% for Advate® , with higher recoveries at lower concentrations. Subgroup analyses by OSA and CSA reagents showed minor variations depending on reagents, but no marked differences between the two products. CSA:OSA ratios based on overall means ranged from 0.99 to 1.17 for Nuwiq® and from 1.01 to 1.17 for Advate® . CONCLUSIONS Both OSAs and CSAs are suitable for the measurement of FVIII:C of Nuwiq® in routine laboratory practice, without the need for a product-specific reference standard

Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin

<p>Abstract</p> <p>Background</p> <p>The incidence of malignant pleural mesothelioma (MPM) is associated with exposure to asbestos, and projections suggest that the yearly number of deaths in Western Europe due to MPM will increase until 2020. Despite progress in chemo- and in multimodality therapy, MPM remains a disease with a poor prognosis. Inducing apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or agonistic monoclonal antibodies which target TRAIL-receptor 1 (TRAIL-R1) or TRAIL-R2 has been thought to be a promising cancer therapy.</p> <p>Results</p> <p>We have compared the sensitivity of 13 MPM cell lines or primary cultures to TRAIL and two fully human agonistic monoclonal antibodies directed to TRAIL-R1 (Mapatumumab) and TRAIL-R2 (Lexatumumab) and examined sensitization of the MPM cell lines to cisplatin-induced by the TRAIL-receptor antibodies. We found that sensitivity of MPM cells to TRAIL, Mapatumumab and Lexatumumab varies largely and is independent of TRAIL-receptor expression. TRAIL-R2 contributes more than TRAIL-R1 to death-receptor mediated apoptosis in MPM cells that express both receptors. The combination of cisplatin with Mapatumumab or Lexatumumab synergistically inhibited the cell growth and enhanced apoptotic death. Furthermore, pre-treatment with cisplatin followed by Mapatumumab or Lexatumumab resulted in significant higher cytotoxic effects as compared to the reverse sequence. Combination-induced cell growth inhibition was significantly abrogated by pre-treatment of the cells with the antioxidant N-acetylcysteine.</p> <p>Conclusion</p> <p>Our results suggest that the sequential administration of cisplatin followed by Mapatumumab or Lexatumumab deserves investigation in the treatment of patients with MPM.</p

p53-Induced Apoptosis Occurs in the Absence of p14(ARF) in Malignant Pleural Mesothelioma

Malignant pleural mesotheliomas (MPMs) are usually wild type for the p53 gene but contain homozygous deletions in the INK4A locus that encodes p14(ARF), an inhibitor of p53-MDM2 interaction. Previous findings suggest that lack of p14(ARF) expression and the presence of SV40 large T antigen (L-Tag) result in p53 inactivation in MPM. We did not detect SV40 L-Tag mRNA in either MPM cell lines or primary cultures, and treatment of p14(ARF)-deficient cells with cisplatin (CDDP) increased both total and phosphorylated p53 and enhanced p53 DNA-binding activity. On incubation with CDDP, levels of positively regulated p53 transcriptional targets p21(WAF), PIG3, MDM2, Bax, and PUMA increased in p14(ARF)-deficient cells, whereas negatively regulated survivin decreased. Significantly, p53-induced apoptosis was activated by CDDP in p14(ARF)-deficient cells, and treatment with p53-specific siRNA rendered them more CDDP-resistant. p53 was also activated by: 1) inhibition of MDM2 (using nutlin-3); 2) transient overexpression of p14(ARF); and 3) targeting of survivin using antisense oligonucleotides. However, it is noteworthy that only survivin downregulation sensitized cells to CDDP-induced apoptosis. These results suggest that p53 is functional in the absence of p14(ARF) in MPM and that targeting of the downstream apoptosis inhibitor survivin can sensitize to CDDP-induced apoptosis

Reactions of N,3-diarylpropiolamides with arenes under superelectrophilic activation: synthesis of 4,4-diaryl-3,4-dihydroquinolin-2(1H)-ones and their derivatives

The reaction of 3-aryl-N-(aryl)propiolamides with arenes in TfOH at room temperature for 0.5 h led to 4,4-diaryl-3,4-dihydroquinolin-2-(1H)-ones in yields of 44–98%. The obtained dihydroquinolinones were further transformed into the corresponding N-acyl or N-formyl derivatives. For the latter, the superelectrophilic activation of the N-formyl group by TfOH in the reaction with benzene resulted in the formation of N-(diphenylmethyl)-substituted dihydroquinolinones

Different methylation characteristics of protein arginine methyltransferase 1 and 3 toward the Ewing Sarcoma protein and a peptide

The multifunctional Ewing Sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, is extensively asymmetrically dimethylated at arginine residues within RGG consensus sequences. Using recombinant proteins we examined whether type I protein arginine methyltransferase (PRMT)1 or 3 is responsible for asymmetric dimethylations of the EWS protein. After in vitro methylation of the EWS protein by GST-PRMT1, we identified 27 dimethylated arginine residues out of 30 potential methylation sites by mass spectrometry-based techniques (MALDI-TOF MS and MS/MS). Thus, PRMT1 recognizes most if not all methylation sites of the EWS protein. With GST-PRMT3, however, only nine dimethylated arginines, located mainly in the C-terminal region of EWS protein, could be assigned, indicating that structural determinants prevent complete methylation. In contrary to previous reports this study also revealed that trypsin is able to cleave after methylated arginines. Pull-down experiments showed that endogenous EWS protein binds efficiently to GST-PRMT1 but less to GST-PRMT3, which is in accordance to the in vitro methylation results. Furthermore, methylation of a peptide containing different methylation sites revealed differences in the site selectivity as well as in the kinetic properties of GST-PRMT1 and GST-PRMT3. Kinetic differences due to an inhibition effect of the methylation inhibitor S-adenosyl-L-homocysteine could be excluded by determining the corresponding K(i) values of the two enzymes and the K(d) values for the methyl donor S-adenosyl-L-methionine. The study demonstrates the strength of MS-based methods for a qualitative and quantitative analysis of enzymic arginine methylation, a posttranslational modification that becomes more and more the object of investigations

Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin-2

<p><b>Copyright information:</b></p><p>Taken from "Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin"</p><p>http://www.molecular-cancer.com/content/6/1/66</p><p>Molecular Cancer 2007;6():66-66.</p><p>Published online 22 Oct 2007</p><p>PMCID:PMC2134932.</p><p></p>cells were cultured with or without 2 μM cisplatin for 24 h. Cells were subsequently left untreated or treated with 10 μg/ml Mapatumumab or Lexatumumab and cell proliferation was determined 72 h later as described under experimental procedures. Absorbance values obtained with untreated cells maintained under identical conditions were taken as 100%. Data represent means of three independent experiments and bars indicate standard deviations. **, < 0.01 and *, < 0.05 compared to cells not treated with anti-TRAIL receptor antibodies

Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin-6

<p><b>Copyright information:</b></p><p>Taken from "Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin"</p><p>http://www.molecular-cancer.com/content/6/1/66</p><p>Molecular Cancer 2007;6():66-66.</p><p>Published online 22 Oct 2007</p><p>PMCID:PMC2134932.</p><p></p>g/ml, 10 μg/ml, 10 μg/ml, respectively) as indicated, either in the presence or absence of NAC (5 mM). Cell proliferation was determined by MTT assay 48 h thereafter. Absorbance values obtained with untreated cells maintained under identical conditions were taken as 100%. Data represent means of at least three independent experiments and bars indicate standard deviations. **, < 0.01 and *, < 0.05 compared to cells treated in the absence of NAC but otherwise maintained under identical conditions

Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin-1

<p><b>Copyright information:</b></p><p>Taken from "Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin"</p><p>http://www.molecular-cancer.com/content/6/1/66</p><p>Molecular Cancer 2007;6():66-66.</p><p>Published online 22 Oct 2007</p><p>PMCID:PMC2134932.</p><p></p>ed by MTT assay 72 h thereafter. Absorbance values obtained with untreated cells maintained under identical conditions were taken as 100%. Representative data of three independent experiments showing similar results are shown here

Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin-0

<p><b>Copyright information:</b></p><p>Taken from "Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin"</p><p>http://www.molecular-cancer.com/content/6/1/66</p><p>Molecular Cancer 2007;6():66-66.</p><p>Published online 22 Oct 2007</p><p>PMCID:PMC2134932.</p><p></p>e columns) were incubated with 100 ng/ml His-TRAIL, and cell proliferation was determined by MTT assay 72 h thereafter. Absorbance values obtained with untreated cells maintained under identical conditions were taken as 100%. Data represent means of three independent experiments and bars indicate standard deviations. **, < 0.01 and *, < 0.05 compared to untreated cells