BACKGROUND.T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS.T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19).RESULTS. SB-mediated genetic transposition and stimulation resulted in 2,200-to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients.CONCLUSIONS. CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach.

Microparticles, which include exosomes, micro-vesicles, apoptotic bodies and apoptotic microparticles, are small (0.05 -3 lm in diameter), membranous vesicles that can contain DNA, RNA, miRNA, intracellular proteins and express extracellular surface markers from the parental cells. They can be secreted from intracellular multivesicular bodies or released from the surface of blebbing membranes. Circulating microparticles are abundant in the plasma of normal individuals and can be derived from circulating blood cells such as platelets, red blood cells and leukocytes as well as from tissue sources, such as endothelial and placental tissues. Elevated levels of microparticles are associated with various diseases such as thrombosis (platelet microparticles), congestive heart failure (endothelial microparticles), breast cancer patients (leukocyte microparticles) and women with preeclampsia (syncytiotrophoblast microparticles). Although microparticles can be detected by microscopy, enzyme-linked immunoassays and functional assays, flow cytometry is the preferred method because of the ability to quantitate (fluorescent bead-or flow rate-based method) and because of polychromatic capabilities. However, standardization of pre-analytical and analytical modus operandi for isolating, enumerating and fluorescent labeling of microparticles remains a challenge. The primary focus of this article is to review the preliminary steps required to optimally study circulating in vivo microparticles which include: 1) centrifugation speed used, 2) quantitation of microparticles before antibody labeling, 3) levels of fluorescence intensity of antibody-labeled microparticles, 4) polychromatic flow cytometric analysis of microparticle sub-populations and 5) use of polyclonal antibodies designed for Western blotting for flow cytometry. These studies determine a roadmap to develop microparticles as biomarkers for a variety of conditions. ' 2010 International Society for Advancement of Cytometry Key terms quantitation of plasma microparticles; polychromatic flow cytometric analysis; Western blotting polyclonal antibodies OVER the years, the quiet waters of flow cytometric analysis of circulating microparticles (MPs) have given way to a deluge of original articles, technical briefs, and critical reviews. However, given the vast scope of work about circulating MPs, it is not possible to cover all the literature. Instead, this review focuses on flow cytometric analysis and applications of fetal-derived MPs. Although other cell-derived MPs such as platelet MPs (PMPs), endothelial MPs (EMPs), and leukocyte MP (LMPs) are not a focus of this review, many of the same principles apply.

Background-The aim of this study was to determine whether DNA-associated micro-particles (MPs) in maternal plasma express fetal-derived human leukocyte antigen-G (HLA-G) or placental alkaline phosphatase (PLAP) and whether the levels differ between women with normotensive pregnancies and preeclampsia.

Autophagy can sustain or kill tumor cells depending upon the context. The mechanism of autophagy-associated cell death has not been well elucidated and autophagy has enhanced or inhibited sensitivity of cancer cells to cytotoxic chemotherapy in different models. ARHI (DIRAS3), an imprinted tumor suppressor gene, is downregulated in 60% of ovarian cancers. In cell culture, re-expression of ARHI induces autophagy and ovarian cancer cell death within 72 h. In xenografts, re-expression of ARHI arrests cell growth and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks, dormancy is broken and xenografts grow promptly. In this study, ARHI-induced ovarian cancer cell death in culture has been found to depend upon autophagy and has been linked to G1 cell-cycle arrest, enhanced reactive oxygen species (ROS) activity, RIP1/RIP3 activation and necrosis. Re-expression of ARHI enhanced the cytotoxic effect of cisplatin in cell culture, increasing caspase-3 activation and PARP cleavage by inhibiting ERK and HER2 activity and downregulating XIAP and Bcl-2. In xenografts, treatment with cisplatin significantly slowed the outgrowth of dormant autophagic cells after reduction of ARHI, but the addition of chloroquine did not further inhibit xenograft outgrowth. Taken together, we have found that autophagy-associated cancer cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells are still vulnerable to cisplatin-based chemotherapy.

Structural and biochemical studies have revealed that procaspases form dimers prior to proteolytic activation. How the two procaspases interact in the dimer is unclear. To study the mechanisms of dimer-dependent caspase activation we used a heterodimeric system so that two caspase molecules can be specifically brought together. Surprisingly, only one caspase partner in the dimer needs to be enzymatically active for caspase processing and activation to occur. Caspase activation is inefficient in the dimer in the absence of intramolecular processing, suggesting that caspase activation is initiated via intramolecular processing. Homodimerization of caspase-8 or caspase-9 leads to the formation of a stable dimeric complex. However, heterodimerization between caspase-8 and caspases-3, -9, or -10 failed to induce stable dimer formation or caspase activation. Our data suggest that the formation of a stable dimeric intermediate initiates caspase activation.