Fiber depolymerization

Depolymerization is, by definition, a crucial process in the reversible assembly of various biopolymers. It may also be an important factor in the pathology of sickle cell disease. If sickle hemoglobin fibers fail to depolymerize fully during passage through the lungs then they will reintroduce aggregates into the systemic circulation and eliminate or shorten the protective delay (nucleation) time for the subsequent growth of fibers. We study how depolymerization depends on the rates of end- and side-depolymerization, kend and kside, which are, respectively, the rates at which fiber length is lost at each end and the rate at which new breaks appear per unit fiber length. We present both an analytic mean field theory and supporting simulations showing that the characteristic fiber depolymerization time View the MathML source depends on both rates, but not on the fiber length L, in a large intermediate regime 1 much less-than ksideL2/kend much less-than (L/d)2, with d the fiber diameter. We present new experimental data which confirms that both mechanisms are important and shows how the rate of side depolymerization depends strongly on the concentration of CO, acting as a proxy for oxygen. Our theory remains rather general and could be applied to the depolymerization of an entire class of linear aggregates, not just sickle hemoglobin fibers

507. Chondroitin Sulfate Proteoglycan 4 (CSPG4)-Redirected T Cells Eliminate Glioblastoma-Derived Neurospheres

Adoptive therapy with chimeric antigen receptor-redirected T cells (CAR-Ts) remains challenging for the treatment of glioblastoma (GBM) because of the heterogeneous expression of targetable tumor antigens, which leads to the selection of antigen-loss variants. In addition, the emerging role of GBM-derived neurospheres (GBM-NS) as a critical cell subset in causing GBM recurrence highlights the need to eradicate these cells to achieve sustained responses. By exploiting a well-established culture system, we generated and expanded GBM-NS from 23 surgical samples, and tested them using flow cytometry for the expression of CSPG4, a membrane bound tumor antigen found to be overexpressed in GBM by mRNA profiling. We observed that 70% of GBM-NS displayed high expression of CSPG4 (from 71% to 99%), 17% moderate-high expression (from 51% to 70%), and 13% moderate-low expression (<50%). Based on these results, we hypothesized that CSPG4-specific CAR-Ts would represent a broadly applicable strategy for the treatment of GBM. We generated CSPG4. CAR-Ts, encoding the 4-1BB endodomain, from 6 healthy donors and tested them against 19 of the 23 generated GBM-NS that robustly grow in vitro. CSPG4.CAR-Ts efficiently eliminated all GBM-NS, with high to moderate-low CSPG4 expression, in co-culture experiments at E:T ratios ranging from 2:5 to 1:5 (0.2±0.5% and 0.6±0.9% residual GBM-NS, respectively). By contrast, GBM-NS continued to grow in the presence of control T cells (60.7±17.6% residual GBM-NS). CSPG4.CAR-Ts, but not control T cells, also rapidly proliferated in response to GBM-NS as evaluated by the CFSE assay. CSPG4. CAR-Ts showed a Th1 cytokine profile in response to GBM-NS, releasing significantly more IFN-γ (3593.8±1718.1 pg/ml/2×10^5 cells) and IL-2 (258.8±153.3 pg/ml/2×10^5 cells) than control T cells (1.8±2.5 and 0.9±1.2 pg/ml/2×10^5 cells, respectively). For the in vivo experiments we compared CSPG4.CAR-Ts encoding CD28, 4-1BB, or CD28-4-1BB co-stimulatory endodomains. Two GBM-NS with moderate-low and high CSPG4 expression, respectively were selected and transduced to express the FFluciferase gene to monitor the tumor growth by in vivo bioluminescence imaging. Both GBM-NS and T cells were intracranially injected in 5 wks old female nude mice. CSPG4.CAR-Ts were efficient in controlling tumor growth of both moderate-low and high CSPG4-expressing GBM-NS. We observed an early eradication of the tumor mass in high-CSPG4 expressing GBM-NS, and a significant improved survival in both mice bearing high or moderate-low CSPG4-expressing GBM-NS. CAR-Ts encoding 4-1BB were significantly more efficient than those encoding CD28 or CD28-4-1BB in prolonging tumor free survival (p=0.04). Our data suggest that CSPG4 is an attractive target for CAR-Ts in GBM and that the strategy we have shown to be effective in mice has the potential to be translated to a clinical setting

Simple Synthesis of Fe3O4@-Activated Carbon from Wastepaper for Dispersive Magnetic Solid-Phase Extraction of Non-Steroidal Anti-Inflammatory Drugs and Their UHPLC–PDA Determination in Human Plasma

In the present society, the recycling and reuse of valuable substances are of utmost im- portance for economic and environmental purposes. At the same time, there is a pressing need to develop new methods to protect the ecosystem from many human activities, including those that have contributed to an ever-increasing presence of pharmaceutical pollutants. In this study, a straightforward approach that applies a magnetic carbon composite for the effective removal of NSAIDs from biological fluids is reported. The composite was produced by recycling wasted hand- kerchiefs, to provide cellulose to the reactive system and then transformed into carbon via calcination at high temperature. The morphological and structural features of the prepared “Fe3O4@-activated carbon” samples were investigated via thermal analysis, X-ray diffraction, Raman spectroscopy, and scanning electron microscopy. Magnetic solid-state extraction was carried out to reveal the adsorption capabilities of the magnetic carbon composite and then combined with UHPLC–PDA for the determination and quantification of five NSAIDs (furprofen, indoprofen, ketoprofen, flurbiprofen, and indomethacin). The method developed herein proved to be fast and accurate. The adsorbent could be reused for up to 10 cycles, without any decrease in performance; thus, it contributes to an intelligent and sustainable economic strategy projected toward minimal waste generation

Canine Melanoma Immunology and Immunotherapy: Relevance of Translational Research

In veterinary oncology, canine melanoma is still a fatal disease for which innovative and long-lasting curative treatments are urgently required. Considering the similarities between canine and human melanoma and the clinical revolution that immunotherapy has instigated in the treatment of human melanoma patients, special attention must be paid to advancements in tumor immunology research in the veterinary field. Herein, we aim to discuss the most relevant knowledge on the immune landscape of canine melanoma and the most promising immunotherapeutic approaches under investigation. Particular attention will be dedicated to anti-cancer vaccination, and, especially, to the encouraging clinical results that we have obtained with DNA vaccines directed against chondroitin sulfate proteoglycan 4 (CSPG4), which is an appealing tumor-associated antigen with a key oncogenic role in both canine and human melanoma. In parallel with advances in therapeutic options, progress in the identification of easily accessible biomarkers to improve the diagnosis and the prognosis of melanoma should be sought, with circulating small extracellular vesicles emerging as strategically relevant players. Translational advances in melanoma management, whether achieved in the human or veterinary fields, may drive improvements with mutual clinical benefits for both human and canine patients; this is where the strength of comparative oncology lies

Mutation analysis of BRAF and KIT in circulating melanoma cells at the single cell level

信州大学博士（医学）・学位論文・平成25年3月31日授与（甲第945号）・境澤 香里BACKGROUND: The availability of molecular-targeted therapies for the treatment of melanoma has emphasised the need to identify mutations in target genes such as BRAF and KIT. Circulating tumour cells (CTC) are present in the peripheral blood of a significant proportion of cancer patients. METHODS: High molecular weight melanoma-associated antigen (HMW-MAA) was used to isolate melanoma cells from peripheral blood as it is selectively expressed at high levels on melanomas. The HMW-MAA-positive cells were isolated using immunomagnetic beads. After removing CD45(+) cells, CTC were identified by staining with MART-1-and gp100-specific antibodies (HMW-MAA(+), CD45(-), MART-1/gp100(+)). Single, isolated CTC were then subjected to BRAF and KIT mutational analysis. RESULTS: CTC (HMW-MAA(+), CD45(-), MART-1/gp100(+)) were isolated from the blood of 11 patients and BRAF and KIT were sequenced in nine and four patients, respectively. The BRAF sequences identified in the CTC were inconsistent with those identified in autologous melanoma tumours in three patients and the KIT sequences were inconsistent in three patients. In addition, polyclonal BRAF mutations were identified in one patient and concomitant mutations in BRAF and KIT were identified in another patient. CONCLUSION: Melanoma cells show clonal heterogeneity. Therefore, CTC genotyping may be crucial for successful molecular-targeted therapy. British Journal of Cancer (2012) 106, 939-946. doi:10.1038/bjc.2012.12 www.bjcancer.com Published online 26 January 2012 (C) 2012 Cancer Research UKArticleBRITISH JOURNAL OF CANCER. 106(5):939-946 (2012)journal articl

MHC class I-related antigen-processing machinery component defects in feline mammary carcinoma.

Defects in HLA class I antigen-processing machinery (APM) component expression and/or function are frequent in human tumors. These defects may provide tumor cells with a mechanism to escape from recognition and destruction by HLA class I antigen-restricted, tumor antigen-specific cytotoxic T cells. However, expression and functional properties of MHC class I antigens and APM components in malignant cells in other animal species have been investigated to a limited extent. However, this information can contribute to our understanding of the mechanisms underlying the association of MHC class I antigen and APM component defects with malignant transformation of cells and to identify animal models to validate targeted therapies to correct these defects. To overcome this limitation in the present study, we have investigated the expression of the catalytic subunits of proteasome (Y, X, and Z) and of immunoproteasome (LMP2, LMP7, and LMP10) as well as of MHC class I heavy chain (HC) in 25 primary feline mammary carcinomas (FMCs) and in 23 matched healthy mammary tissues. We found a reduced expression of MHC class I HC and of LMP2 and LMP7 in tumors compared with normal tissues. Concordantly, proteasomal cleavage specificities in extracts from FMCs were different from those in healthy tissues. In addition, correlation analysis showed that LMP2 and LMP7 were concordantly expressed in FMCs, and their expression was significantly correlated with that of MHC class I HC. The abnormalities we have found in the APM in FMCs may cause a defective processing of some tumor antigens