In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110␦ signaling in multiple myeloma ( IntroductionThe bone marrow (BM) microenvironment plays a crucial role in pathogenesis of multiple myeloma (MM) by promoting cell proliferation, survival, migration, and drug resistance. [1][2][3][4] The PI3K/AKT pathway mediates growth and drug resistance in MM cells and also plays a significant role in autophagy. 5,6 PI3K is activated via upstream tyrosine kinase-associated receptors for growth factors, cytokines, antigens, and costimulatory molecules. It in turn activates AKT, which mediates cell proliferation, cell cycle, apoptosis, and autophagy. 7 Class IA PI3K consists of 5 isoforms of regulatory subunits (p85␣, p50␣, p55␣, p85␤, and p55␥), which interact with class IA isoforms. Class IA PI3K is composed of p110␣, -␤, and -␦ isoforms. 8 Among the 8 distinct mammalian isoforms of PI3K, class I PI3Ks are responsible for Akt activation. Importantly, p110␦ is expressed in many cancers, including colon and bladder carcinoma, glioblastoma, and acute myeloid leukemia blasts. 9,10 In the current study, we demonstrate high expression of p110␦ in patient MM cells. Previous studies have shown that CAL-101, a potent and selective p110␦ inhibitor, has broad antitumor activity against cancer cells of hematologic origin. 11,12 Moreover, inhibition of p110␦ induces cleavage of caspases and LC3, consistent with apoptotic and autophagic cell death, respectively. Here we show that p110␦ blockade with CAL-101, a potent and selective p110␦ inhibitor, inhibits MM cell growth even in the presence of interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or bone marrow stromal cells (BMSCs), associated with decreased phosphorylation of AKT and P70S6k. We also confirmed inhibition of human MM cell growth triggered by p110␦ inhibition in our xenograft mouse models of human MM. These studies therefore show that small molecule inhibitors of p110␦ trigger significant anti-MM cytotoxicity both in vitro and in vivo, providing the framework for their clinical evaluation to improve patient outcome in MM. Methods Materialsp110␦ inhibitor CAL-101 and IC488743 were provided by Calistoga Pharmaceuticals. CAL-101 was dissolved in dimethyl sulphoxide at 10mM and stored at Ϫ20°C for in vitro study. IC488743 was dissolved in 1% carboxyl methylcellulose/0.5% Tween 80 and stored at 4°C for in vivo study. Recombinant human p110␣, ␤, ␥, and ␦ were reconstituted with sterile phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin. Bortezomib was provided by Millennium Pharmaceuticals. 3-Methyladenine was purchased from Sigma-Aldrich. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. Cell culture Dex 1460BLOOD, 2 SEPTEMBER 2010 ⅐ VOLUME 116, NUMBER 9For personal use only. on March 28, 2019. by guest www.bloodjournal.org From Germany). LB human MM ce...

Reduction of immunocompetent T cells followed by prolonged lymphopenia may be associated with poor prognosis in elderly septic patients.

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Technologies that delivery antigen-encoded plasmid DNA (pDNA) to antigen presenting cell and their immune-activation are required for the success of DNA vaccines. Here we report on an artificial nanoparticle that can achieve these; a multifunctional envelope-type nanodevice modified with KALA, a peptide that forms α-helical structure at physiological pH (KALA-MEND). KALA modification and the removal of the CpG-motifs from the pDNA synergistically boosted transfection efficacy. In parallel, transfection with the KALA-MEND enhances the production of multiple cytokines and chemokines and co-stimulatory molecules via the Toll-like receptor 9-independent manner. Endosome-fusogenic lipid envelops and a long length of pDNA are essential for this immune stimulation. Furthermore, cytoplasmic dsDNA sensors that are related to the STING/TBK1 pathway and inflammasome are involved in IFN-β and IL-1β production, respectively. Consequently, the robust induction of antigen-specific cytotoxic T-lymphoma activity and the resulting prophylactic and therapeutic anti-tumor effect was observed in mice that had been immunized with bone marrow-derived dendritic cells ex vivo transfected with antigen-encoding pDNA. Collectively, the KALA-MEND possesses dual functions; gene transfection system and immune-stimulative adjuvant, those are both necessary for the successful DNA vaccine.

A rapid and specific method for simultaneous quantitation of organophosphates [fenitrothion (MEP), malathion (MAL), and phenthoate (PAP)], glyphosate (GLYP), and glufosinate (GLUF) in human serum and urine by gas chromatography-mass spectrometry (GC-MS) was validated. All targeted compounds, together with the internal standards [fenitrothion-d 6 (MEP-d 6 ) and DL-2-amino-3-phosphonopropionic acid (APPA)], were extracted from the serum and urine with a mixed-mode C-C 18 monolithic spin column. Extracted samples were derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) containing 1% tertbutyldimethylchlorosilane (TBDMCS). Linear calibration curves for serum and urine spanned the ranges of 0.1-10.0 lg/ml for organophosphates, 10-100.0 lg/ml for GLYP, and 5-100.0 lg/ml for GLUF. Detection limits for serum and urine were 10 and 5 lg/ml for GLYP and GLUF, respectively, and quantitation limits for serum and urine were 10 and 5 lg/ml for GLYP and GLUF, respectively. Both detection and quantitation limits for serum and urine were 0.1 lg/ml for organophosphates. The intraassay and interassay accuracy and precision (expressed as relative standard deviation, %RSD) evaluated for all compounds were within 96.5-109.5% and 5.9-13.1%, respectively. The recovery of organophosphates from serum and urine, spiked at concentrations of 0.1, 1.5, 5, and 9 lg/ml, ranged from 58.8 to 90.8%. Recovery of GLYP and GLUF from serum and urine, spiked at concentrations of 10 (serum) or 5 (urine), 15, 50, and 90 lg/ml, ranged from 2.6 to 6.9%. This method had a chromatographic run time of 16.0 min. The validated method was successfully applied to a clinical case of GLYP poisoning.

Methods for quantitative analysis of long distance lymphatic transport of nanoparticles in live animals are yet to be established. We established a mouse model for analysis of time-dependent transport just beneath the abdominal skin to investigate lymph node-to-lymph node trafficking by in vivo imaging. For this purpose, popliteal lymph nodes (PLNs) as well as efferent and afferent lymphatic vessels, marginal veins, and feeding blood vessels were surgically resected to change the lymphatic flow from footpad injections. Using this model, we observed a novel lymphatic flow from the footpad to the proper axillary lymph node (ALN) via the inguinal lymph node (ILN). This drainage pathway was maintained over 12 weeks. Time-dependent transportation of 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide-labelled liposomes from the footpad to the ILN was successfully quantified by an in vivo imaging system. Moreover, congestion and development of a new collateral lymphatic route was visualised under a lymphedema status. Histological analysis of abdominal skin tissues of this model revealed that PLN resection had no effect on the abdominal lymphatic system between the ILN and ALN. These data indicate that this model might be useful to clarify the mechanisms of lymphedema and study direct transportation of lymph or other substances between lymph nodes.

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