Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene RasV12 (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

ObjectiveImmune dysregulation during sepsis is poorly understood, however, lymphocyte apoptosis has been shown to correlate with poor outcomes in septic patients. The inflammasome, a molecular complex which includes caspase-1, is essential to the innate immune response to infection and also important in sepsis induced apoptosis. Our group has recently demonstrated that endotoxin-stimulated monocytes release microvesicles (MVs) containing caspase-1 that are capable of inducing apoptosis. We sought to determine if MVs containing caspase-1 are being released into the blood during human sepsis and induce apoptosis..DesignSingle-center cohort studyMeasurements50 critically ill patients were screened within 24 hours of admission to the intensive care unit and classified as either a septic or a critically ill control. Circulatory MVs were isolated and analyzed for the presence of caspase-1 and the ability to induce lymphocyte apoptosis. Patients remaining in the ICU for 48 hours had repeated measurement of caspase-1 activity on ICU day 3.Main ResultsSeptic patients had higher microvesicular caspase-1 activity 0.05 (0.04, 0.07) AFU versus 0.0 AFU (0, 0.02) (p<0.001) on day 1 and this persisted on day 3, 0.12 (0.1, 0.2) versus 0.02 (0, 0.1) (p<0.001). MVs isolated from septic patients on day 1 were able to induce apoptosis in healthy donor lymphocytes compared with critically ill control patients (17.8±9.2% versus 4.3±2.6% apoptotic cells, p<0.001) and depletion of MVs greatly diminished this apoptotic signal. Inhibition of caspase-1 or the disruption of MV integrity abolished the ability to induce apoptosis.ConclusionThese findings suggest that microvesicular caspase-1 is important in the host response to sepsis, at least in part, via its ability to induce lymphocyte apoptosis. The ability of microvesicles to induce apoptosis requires active caspase-1 and intact microvesicles.

Purpose: Activation of Toll-like receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors. Here, we wished to test the effect of TLR7/8 activation on monocyte Fcγ receptor (FcγR) function, as they are critical mediators of antibody therapy.Experimental Design: The effect of the TLR7/8 agonist R-848 on cytokine production and antibodydependent cellular cytotoxicity by human peripheral blood monocytes was tested. Affymetrix microarrays were done to examine genomewide transcriptional responses of monocytes to R-848 and Western blots were done to measure protein levels of FcγR. Murine bone marrow-derived macrophages from WT and knockout mice were examined to determine the downstream pathway involved with regulating FcγR expression. The efficacy of R-848 as an adjuvant for antibody therapy was tested using a CT26-HER2/neu solid tumor model.Results: Overnight incubation with R-848 increased FcγR-mediated cytokine production and antibodydependent cellular cytotoxicity in human peripheral blood monocytes. Expression of FcγRI, FcγRIIa, and the common γ-subunit was increased. Surprisingly, expression of the inhibitory FcγRIIb was almost completely abolished. In bone marrow-derived macrophage, this required TLR7 and MyD88, as R-848 did not increase expression of the γ-subunit in TLR7 −/− nor MyD88 Monocyte Fcγ receptors (FcγR) mediate clearance of IgG-immune complexes and IgG-coated tumor targets. Binding of IgG complexes to FcγR results in receptor clustering, which activates downstream events such as phagocytosis (1), release of reactive oxygen species (2), and cytokine production (3).The strength of FcγR response is largely determined by the ratio of activating (FcγRI, FcγRIIa, FcγRIII, and the γ-subunit) to inhibitory (FcγRIIb) receptors, as mice genetically deleted for FcγRIIb show markedly enhanced antibody-mediated tumor clearance in vivo (4). Conversely, mice lacking the common γ-subunit show very poor antibody-dependent cytotoxicity as mice do not express the γ-subunit-independent FcγRIIa (5). It has also been shown that Toll-like receptor (TLR) activation can enhance FcγR expression and function. For example, the TLR4 ligand lipopolysaccharide has been shown to increase FcγR-mediated phagocytosis (6) and tumor cell lysis (7). Unmethylated DNA (CpG oligonucleotides), which activates TLR9, has also proven effective, enhancing antibody-dependent cellular cytotoxicity (ADCC) against tumors (8).Agonists of TLR7 and TLR8 have come to light as an effective means of enhancing immune responses. The TLR7 agonist imiquimod has been shown in vivo to reduce the growth of MC-26 tumor cells (9), an effect abolished by blocking IFN-α. Both TLR7 and TLR7/8 agonists show antitumor (10) and antiviral (11) activities. Their major mode of action seems to be induction of cytokine production, leading to stronger proinflammatory responses (12).Here, we have studied the effects of the TLR7/8 agonist R-848 on human monocytes within the context of FcγR expression and function. Results sho...