Intradermal Vaccinations With RNA Coding for TAA Generate CD8+ and CD4+ Immune Responses and Induce Clinical Benefit in Vaccinated Patients

The aim of this phase I/II nonrandomized trial was to assess feasibility, safety as well as immunological and clinical responses of a mRNA-based vaccination in patients with stage IV renal cell cancer using granulocyte-macrophage colony stimulating factor (GM-CSF) as adjuvant. Intradermal injections of in vitro transcribed naked mRNA, which was generated using plasmids coding for the tumor-associated antigens mucin 1(MUC1), carcinoembryonic (CEA), human epidermal growth factor receptor 2 (Her-2/neu), telomerase, survivin, and melanoma-associated antigen 1 (MAGE-A1) were performed in 30 enrolled patients. In the first 14 patients (cohort A) vaccinations were administered on days 0, 14, 28, and 42 (20 µg/antigen) while in the consecutive 16 patients (cohort B) an intensified protocol consisting of injections at days 0–3, 7–10, 28, and 42 (50 µg/antigen) was used. In both cohorts, after this induction period, vaccinations were repeated monthly until tumor progression analyzed by Response Evaluation Criteria In Solid Tumors criteria (RECIST). Vaccinations were well tolerated with no severe side effects and induced clinical responses [six stable diseases (SD) and one partial response in cohort A and nine SD in cohort B]. In cohort A, 35.7% survived 4 years (median survival 24 months) compared to 31.25% in cohort B (median survival 29 months). Induction of CD4⁺ and CD8⁺ T cell responses was shown for several tumor-associated antigens (TAA) using interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and Cr-release assays.     

Labeling Human Mesenchymal Stem Cells with Fluorescent Contrast Agents: the Biological Impact

Purpose  

This study aims to determine the effect of human mesenchymal stem cell (hMSC) labeling with the fluorescent dye DiD and the iron oxide nanoparticle ferucarbotran on chondrogenesis.     

Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein

We show that translation initiation factor EF-Tu plays a second important role in cell shape maintenance in the bacterium Bacillus subtilis. EF-Tu localizes in a helical pattern underneath the cell membrane and colocalizes with MreB, an actin-like cytoskeletal element setting up rod cell shape. The localization of MreB and of EF-Tu is interdependent, but in contrast to the dynamic MreB filaments, EF-Tu structures are more static and may serve as tracks for MreB filaments. In agreement with this idea, EF-Tu and MreB interact in vivo and in vitro. Lowering of the EF-Tu levels had a minor effect on translation but a strong effect on cell shape and on the localization of MreB, and blocking of the function of EF-Tu in translation did not interfere with the localization of MreB, showing that, directly or indirectly, EF-Tu affects the cytoskeletal MreB structure and thus serves two important functions in a bacterium.     

Vimentin cleavage in end-stage renal disease is not related to apoptosis

Anti-vimentin auto-antibodies contribute to chronic allograft nephropathy. They exist in sera of end-stage renal disease patients on hemodialysis (ESRD) already before renal transplantation. We found recently that a 49 kDa vimentin fragment is increased in lymphocytes of ESRD patients which is presented on the cell surface. In vitro studies showed that such a fragment is formed during apoptosis by active caspase-3. We hypothesized that vimentin degradation in leukocytes of ESRD patients correlates to caspase-3 activation in vivo. Lymphocytes and monocytes were isolated from ESRD patients and from healthy volunteers and analyzed for vimentin expression and caspase-3 activation. In addition, apoptosis was induced in vitro and quantified by flow cytometry. ESRD monocytes have shown only the full length 60 kDa vimentin isoform. ESRD lymphocytes, however, showed in addition a strongly increased expression of the 49 kDa vimentin in all samples. Caspase-3 activation was found in 60% of ESRD lymphocytes and 66% of ESRD monocytes but not in healthy volunteers. UV-mediated induction of apoptosis was not associated with vimentin degradation. These experiments could confirm increased vimentin degradation in ESRD lymphocytes. However, we could not validate any correlation to apoptosis.     

Continuously moving table time‐of‐flight angiography of the peripheral veins

Time‐of‐flight (TOF) MR angiography allows for noninvasive vessel imaging. To overcome the limited volumetric coverage of standard TOF techniques, the aim of this study was to investigate the combination of TOF and continuously moving table (CMT) acquisitions for peripheral vein imaging based on image subtraction. Two acquisition strategies are presented: a simple two‐step method based on 2‐fold CMT acquisition and an advanced one‐step method requiring only one continuous scan. Image quality of both CMT TOF techniques was evaluated by semiquantitative image grading and by signal‐to‐noise ratio and contrast‐to‐noise ratio analysis for veins of the upper and lower leg in 10 healthy volunteers. Results were compared to a standard stationary two‐dimensional (2D) TOF multistation acquisition. Image grading revealed good image quality for both CMT TOF methods, thereby confirming the feasibility of axial 2D CMT TOF to assess the veins of the lower extremities during a single scan. Quantitative evaluation showed no significant difference in signal‐to‐noise ratio and contrast‐to‐noise ratio compared to the stationary experiment. Additional measurements in three patients with postthrombotic changes and varicosities demonstrated the clinical applicability of the presented methods. CMT TOF provides promising results and permits the detection of various pathologic changes of the venous system. Magn Reson Med 63:1219–1229, 2010. © 2010 Wiley‐Liss, Inc.     

A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN

The PI 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10), one of the most important tumor suppressors, must associate with the plasma membrane to maintain appropriate steady-state levels of phosphatidylinositol 3,4,5-triphosphate. Yet the mechanism of membrane binding has received little attention and the key determinants that regulate localization, a phosphatidylinositol 4,5-bisphosphate (PIP₂) binding motif and a cluster of phosphorylated C-terminal residues, were not included in the crystal structure. We report that membrane binding requires PIP₂ and show that phosphorylation regulates an intramolecular interaction. A truncated version of the enzyme, PTEN_1–351, bound strongly to the membrane, an effect that was reversed by co-expression of the remainder of the molecule, PTEN_352–403. The separate fragments associated in vitro, an interaction dependent on phosphorylation of the C-terminal cluster, a portion of the PIP₂ binding motif, integrity of the phosphatase domain, and the CBR3 loop. Our investigation provides direct evidence for a model in which PTEN switches between open and closed states and phosphorylation favors the closed conformation, thereby regulating localization and function. Small molecules targeting these interactions could potentially serve as therapeutic agents in antagonizing Ras or PI3K-driven tumors. The study also stresses the importance of determining the structure of the native enzyme.