Reduced cytotoxicity by mutation of lysine 590 of Pseudomonas exotoxin can be restored in an optimized, lysine-free immunotoxin.

Immunotoxins, which are fusion proteins of an antibody fragment and a fragment of a bacterial or a plant toxin, induce apoptosis in target cells by inhibition of protein synthesis. ADP-ribosylating toxins often have few lysine residues in their catalytic domain. As they are the target for ubiquitination, the low number of lysines possibly prevents ubiquitin-dependent degradation of the toxin in the cytosol. To reduce this potential degradation, we aimed to generate a lysine-free (noK), Pseudomonas exotoxin (PE)-based immunotoxin. The new generation 24 kDa PE, which lacks all but the furin-cleavage site of domain II, was mutated at lysine 590 (K590) and at K606 in a CD22-targeting immunotoxin and activity was determined against various B cell malignancies in vitro and in vivo. On average, K590 mutated to arginine (R) reduced cytotoxicity by 1.3-fold and K606R enhanced cytotoxicity by 1.3-fold compared to wild type (wt). Mutating K590 to histidine or deleting K590 did not prevent this loss in cytotoxicity. Neither stability nor internalization rate of K590R could explain reduced cytotoxicity. These results highlight the relevance of lysine 590 for PE intoxication. In line with in vitro results, the K606R mutant was more than 1.8-fold more active than the other variants in vivo suggesting that this single mutation may be beneficial when targeting CD22-positive malignancies. Finally, reduced cytotoxicity by K590R was compensated for by K606R and the resulting lysine-free variant achieved wt-like activity in vitro and in vivo. Thus, PE24-noK may represent a promising candidate for down-stream applications that would interfere with lysines.

Usability and Suitability of the Omics-Integrating Analysis Platform tranSMART for Translational Research and Education.

BACKGROUND: Platforms like tranSMART assist researchers in analyzing clinical and corresponding omics data. Usability is an important, yet often overlooked, factor affecting the adoption and meaningful use. Analyses on the specific needs of translational researchers and considerations about the application of such platforms for education are rare. OBJECTIVES: The aim of this study was to test whether tranSMART can be used in education and how well medical students and professional researchers can handle it; to identify which kind of translational researchers-in terms of skills, experienced limitations, and available data-can take advantage of tranSMART; and to evaluate the usability and to generate recommendations for improvements. METHODS: An online-based test has been done by medical students (N = 109) and researchers (N = 26). The test comprised 13 tasks in the context of four typical research scenarios based on experimental and clinical data. A web questionnaire was provided to identify both the needs and the conditions of research as well as to evaluate the system's usability based on the "System Usability Scale" (SUS). RESULTS: Students and researchers were able to handle tranSMART well and coped with most scenarios: cohort identification, data exploration, hypothesis generation, and hypothesis validation were answered with a rate of correctness between 82 and 100%. Of the total, 72.2% of the teaching researchers considered tranSMART suitable for their lessons and 84.6% of the researchers considered the platform useful for their daily work; 65.4% of the researchers named the nonavailability of a platform like tranSMART as a restriction on their research. The usability was rated "acceptable" with a SUS of 70.8. CONCLUSION: tranSMART is potentially suitable for education purposes and fits most of the needs of translational researchers. Improvements are needed on the presentation of analysis results and on the guidance of users through the analysis, especially to ensure the compliance of the analysis with the requirements of statistical testing.

Efficacy of aflibercept (EYLEA®) on inhibition of human VEGF in vitro.

INTRODUCTION: Pathological formation of blood vessels plays a key role in the growth and metastasis of tumors and also in several serious ophthalmological diseases such as wet age-related macular degeneration (AMD) or diabetic retinopathy. In AMD treatment, aflibercept (tradename EYLEA®) is used to deactivate the underlying pathological neovascularisation. Aflibercept is a recombinant fusion protein which binds to vascular endothelial growth factor (VEGF) receptors, thereby inhibiting VEGF pathway activation. VEGF is one of the most important angiogenesis factors. OBJECTIVE: This analysis investigates lasting efficacy of aflibercept in vitro for later application as therapeutic agent against macular degeneration (AMD). MATERIAL AND METHODS: VEGF-ELISA assays were performed to investigate binding affinities at different aflibercept concentrations. The impact of VEGF on the proliferation of human umbilical vein endothelial cells (HUVEC) was investigated using proliferation assays. Moreover, time-dependent kinetic studies were performed to analyze different aflibercept storage durations with regard to its inhibitory capabilities on human VEGF. RESULTS AND CONCLUSION: Our results reveal that aflibercept significantly lowers the amount of unbound VEGF as well as the proliferation rate of HUVEC. Moreover, in contrast to specifications given by the manufacturer, aflibercept retains its full inhibitory effect up to at least 120h after transference from the original vial into the injection syringe.

Structural proteins of Kaposi's sarcoma-associated herpesvirus antagonize p53-mediated apoptosis.

The tumor suppressor p53 is a central regulatory molecule of apoptosis and is commonly mutated in tumors. Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies express wild-type p53. Accordingly, KSHV encodes proteins that counteract the cell death-inducing effects of p53. Here, the effects of all KSHV genes on the p53 signaling pathway were systematically analyzed using the reversely transfected cell microarray technology. With this approach we detected eight KSHV-encoded genes with potent p53 inhibiting activity in addition to the previously described inhibitory effects of KSHV genes ORF50, K10 and K10.5. Interestingly, the three most potent newly identified inhibitors were KSHV structural proteins, namely ORF22 (glycoprotein H), ORF25 (major capsid protein) and ORF64 (tegument protein). Validation of these results with a classical transfection approach showed that these proteins inhibited p53 signaling in a dose-dependent manner and that this effect could be reversed by small interfering RNA-mediated knockdown of the respective viral gene. All three genes inhibited p53-mediated apoptosis in response to Nutlin-3 treatment in non-infected and KSHV-infected cells. Addressing putative mechanisms, we could show that these proteins could also inhibit the transactivation of the promoters of apoptotic mediators of p53 such as BAX and PIG3. Altogether, we demonstrate for the first time that structural proteins of KSHV can counteract p53-induced apoptosis. These proteins are expressed in the late lytic phase of the viral life cycle and are incorporated into the KSHV virion. Accordingly, these genes may inhibit cell death in the productive and in the early entrance phase of KSHV infection.

IFN-γ and TNF-α-induced GBP-1 inhibits epithelial cell proliferation through suppression of ß-catenin/TCF signaling.

Proinflammatory cytokines induce guanylate-binding protein 1 (GBP-1) protein expression in intestinal epithelial tissues. GBP-1 has been described as influencing a number of cellular processes important for epithelial homeostasis, including cell proliferation. However, many questions remain as to the role of GBP-1 in intestinal mucosal homeostasis. We therefore sought to investigate the function of proinflammatory cytokine-induced GBP-1 during intestinal epithelial cell proliferation. Through the use of complementary GBP-1 overexpression and small interfering RNA-mediated knockdown studies, we now show that GBP-1 acts to inhibit pro-mitogenic ß-catenin/T cell factor (TCF) signaling. Interestingly, proinflammatory cytokine-induced GBP-1 was found to be a potent suppressor of ß-catenin protein levels and ß-catenin serine 552 phosphorylation. Neither glycogen synthase kinase 3ß nor proteasomal inhibition alleviated GBP-1-mediated suppression of cell proliferation or ß-catenin/TCF signaling, indicating a non-canonical mechanism of ß-catenin inhibition. Together, these data show that cytokine-induced GBP-1 retards cell proliferation by forming a negative feedback loop that suppresses ß-catenin/TCF signaling.