Isolation of single cells from individual mouse ovaries for flow cytometry and functional analysis.

Here, we present a validated workflow to isolate sufficient viable single ovary cells from a single mouse without the need to pool from several mice. We provide steps essential for estrous staging, ovary harvesting and dissociation, ovary cell staining, data collection, and analysis. Our approach allows the use of these single-cell suspensions for flow sorting, flow cytometry analysis, or functional in vitro assays. Importantly, our protocol is designed to maximize the isolation of immune cells, including T cell subsets.

The KIR2DL1 intermediate upstream element participates in gene activation.

The human KIR genes encode a family of class I MHC receptors that are expressed on subsets of NK cells. The expression of KIR proteins is controlled by a stochastic process, and competition between sense and antisense promoter elements has been suggested to program the variegated expression of these genes. Previous studies have demonstrated distinct roles of distal, intermediate, and proximal sense promoter/enhancer elements in gene activation and expression. Conversely, proximal and intronic antisense promoter transcripts have been associated with gene silencing at different stages of NK cell development. In the current study, we examine the effect of intermediate promoter deletion on KIR2DL1 expression in the YTS cell line. Homozygous deletion of the KIR2DL1 intermediate element did not affect proximal promoter activity but resulted in increased detection of upstream transcripts. No significant changes in alternative mRNA splicing or expression levels of KIR2DL1 protein were observed. However, intermediate element deletion was associated with a reduced frequency of gene activation by 5-azacytidine. Taken together, these results indicate that the intermediate element is not an enhancer required for KIR expression; however, it is required for the efficient activation of the gene.

Tag-on-Demand: exploiting amber codon suppression technology for the enrichment of high-expressing membrane protein cell lines.

Membrane proteins play key roles in the evolution of numerous diseases and as a result have become the most dominant class of targets for therapeutic intervention. However, their poor expression and detection oftentimes prohibit drug discovery and screening efforts. Herein, we have developed an approach, named 'Tag-on-Demand' that exploits amber suppression to control the expression of 'tagged' membrane proteins for detection and selections, yet can be turned off for expression of the protein in its native form. Utilizing an engineered Chinese hamster ovary cell line capable of efficient amber suppression, we evaluated the expression of a diverse panel of model membrane proteins and demonstrated the enrichment of cells with improved expression profiles, where ~200-800% improvement in total protein expression levels were observed over pre-sorted populations after a single round of fluorescence-activated cell sorting. Furthermore, results were most striking for the typically difficult-to-express G protein-coupled receptor, CXCR2, where ~2.5-fold improvement in surface expression was observed. We anticipate that the Tag-on-Demand approach will be suitable not only for membrane protein cell line development but also for the development of intracellular and secreted protein cell lines in expression systems for which amber suppression technology exists, including bacterial, yeast, insect and cell-free expression systems.

Fusion toxin BLyS-gelonin inhibits growth of malignant human B cell lines in vitro and in vivo.

B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. The biological activity of BLyS is mediated by three cell surface receptors: BR3/BAFF-R, TACI and BCMA. The expression of these receptors is highly restricted to B cells, both normal and malignant. A BLyS-gelonin fusion toxin (BLyS-gel) was generated consisting of the recombinant plant-derived toxin gelonin fused to the N-terminus of BLyS and tested against a large and diverse panel of B-NHL cell lines. Interestingly, B-NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with low picomolar EC(50) values. BLyS receptor expression did not guarantee sensitivity to BLyS-gel, even though the construct was internalized by both sensitive and resistant cells. Resistance to BLyS-gel could be overcome by treatment with the endosomotropic drug chloroquine, suggesting BLyS-gel may become trapped within endosomal/lysosomal compartments in resistant cells. BLyS-gel induced cell death was caspase-independent and shown to be at least partially mediated by the "ribotoxic stress response." This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment was shown to localize to sites of disease, rapidly reduce tumor burden, and significantly prolong survival in xenograft mouse models of disseminated BCP-ALL, DLBCL, and MCL. Together, these findings suggest BLyS has significant potential as a targeting ligand for the delivery of cytotoxic "payloads" to malignant B cells.

Mapatumumab and lexatumumab induce apoptosis in TRAIL-R1 and TRAIL-R2 antibody-resistant NSCLC cell lines when treated in combination with bortezomib.

Mapatumumab and lexatumumab are fully human monoclonal antibodies that bind and activate human tumor necrosis factor-related apoptosis-inducing ligand receptors 1 and 2, respectively. These antibodies induce apoptosis in various tumor cell types, although the degree of sensitivity can vary from highly sensitive to completely resistant. Importantly, tumor cells that are partially or completely resistant to mapatumumab or lexatumumab can often be sensitized when treated in combination with chemotherapeutic drugs. In this regard, the proteasome inhibitor bortezomib has recently shown synergistic activity against established lymphoma cell lines and primary lymphomas when combined with mapatumumab and lexatumumab. Here, we report similar findings using a panel of human non-small cell lung cancer (NSCLC) cell lines. Specifically, we show that bortezomib rapidly induces sensitivity to mapatumumab and lexatumumab in NSCLC cell lines that are completely resistant to antibody alone and that bortezomib concentrations as low as 25 nmol/L sensitize NSCLC cells to the antibodies. Furthermore, bortezomib at the tested concentration has minimal effect on its own, indicating the combination generates synergistic cytotoxicity. Combination treatment induces activation of the caspase cascade and the effect of the combination is caspase dependent. Bortezomib treatment increases the intracellular levels of several important apoptosis regulators that may mediate enhanced sensitivity to mapatumumab and lexatumumab. These results suggest future evaluation of mapatumumab or lexatumumab in combination with bortezomib is warranted in NSCLC patients.

Albugranin, a recombinant human granulocyte colony stimulating factor (G-CSF) genetically fused to recombinant human albumin induces prolonged myelopoietic effects in mice and monkeys.

PURPOSE: Albugranin fusion protein is recombinant granulocyte colony stimulating factor (rG-CSF) genetically fused at its N-terminus to the C-terminus of recombinant serum human albumin and is expected to have a relatively long half-life compared with rG-CSF alone. In this study, the pharmacodynamics and pharmacokinetics of Albugranin were evaluated in BDF1 mice and cynomolgus monkeys. METHODS: Single doses of Albugranin (0.25-5 mg/kg) or Filgrastim (methionyl rG-CSF, 0.25, or 1.25 mg/kg) were administered subcutaneously (SC) to mice and multiple doses of Albugranin (25-100 microg/kg every 4 or 7 days) or Filgrastim (5 microg/kg daily) were administered SC for 14 days to monkeys for hematologic evaluation. For pharmacokinetics studies, mice were injected intravenously (IV) or SC with single doses of Albugranin (0.25-1.25 mg/kg) or Filgrastim (0.25 mg/ kg) and monkeys were injected SC with multiple doses of Albugranin (100-1,000 microg/kg once weekly for 5 weeks). Plasma levels of Albugranin and Filgrastim were measured by enzyme-linked immunosorbent assay. RESULTS: In mice, administration of Albugranin effectively increased the number of peripheral granulocytes and mobilized hematopoietic progenitor cells for up to 5 days. The magnitude and duration of this effect were dose-dependent. In contrast, administration of Filgrastim resulted in a small increase in both cell types on day 1 only. Albugranin administered to cynomolgus monkeys caused an increase in peripheral neutrophils, with a less prominent increase in peripheral monocytes. Albugranin-induced neutrophilia peaked 24 h following each dose administration. Administration of Filgrastim daily in monkeys resulted in moderate increases in neutrophils that were maximal on days 8-12 during the course of treatment. Compared with Filgrastim, Albugranin had a longer terminal half-life (t(1/2,term)) and mean residence time (MRT), and slower clearance (CL/F) in mice. The t(1/2,term), MRT, and CL/F of Albugranin following SC administration to BDF1 mice were 5.6-5.7 h, 16.7-20.7 h, and 6.37-12.2 mL/h/kg, respectively, compared with 2.54 h, 4.9 h, and 164 mL/h/kg, respectively for Filgrastim. In cynomolgus monkeys, the corresponding values of t(1/2,term), MRT, and CL/F for Albugranin were 7.73-133 h, 19.4-27.3 h, and 7.90-27.5 mL/h/kg, respectively, for doses of 100-1000 microg/kg. An exposure-response relationship that could be empirically described with a simple Emax model with baseline was found between day 15 absolute neutrophil count and area under the curve following the first dose in cynomolgus monkeys. CONCLUSION: The sustained activity of Albugranin in mice and monkeys demonstrated in these studies suggests that this agent could be given less frequently than Filgrastim to achieve similar therapeutic effects in patients.

TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator.

DR3 is a death domain-containing receptor that is upregulated during T cell activation and whose overexpression induces apoptosis and NF-kappaB activation in cell lines. Here we show that an endothelial cell-derived TNF-like factor, TL1A, is a ligand for DR3 and decoy receptor TR6/DcR3 and that its expression is inducible by TNF and IL-1alpha. TL1A induces NF-kappaB activation and apoptosis in DR3-expressing cell lines, while TR6-Fc protein antagonizes these signaling events. Interestingly, in T cells, TL1A acts as a costimulator that increases IL-2 responsiveness and secretion of proinflammatory cytokines both in vitro and in vivo. Our data suggest that interaction of TL1A with DR3 promotes T cell expansion during an immune response, whereas TR6 has an opposing effect.