Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.13.12.7 (firefly luciferase)
 2,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Z-100 is an arabinomannan extracted from Mycobacterium tuberculosis that has various immunomodulatory activities, such as the induction of interleukin 12, interferon gamma (IFN-gamma) and beta-chemokines. The effects of Z-100 on human immunodeficiency virus type 1 (HIV-1) replication in human monocyte-derived macrophages (MDMs) are investigated in this paper. In MDMs, Z-100 markedly suppressed the replication of not only macrophage-tropic (M-tropic) HIV-1 strain (HIV-1JR-CSF), but also HIV-1 pseudotypes that possessed amphotropic Moloney murine leukemia virus or vesicular stomatitis virus G envelopes. Z-100 was found to inhibit HIV-1 expression, even when added 24 h after infection. In addition, it substantially inhibited the expression of the pNL43lucDeltaenv vector (in which the env gene is defective and the nef gene is replaced with the firefly luciferase gene) when this vector was transfected directly into MDMs. These findings suggest that Z-100 inhibits virus replication, mainly at HIV-1 transcription. However, Z-100 also downregulated expression of the cell surface receptors CD4 and CCR5 in MDMs, suggesting some inhibitory effect on HIV-1 entry. Further experiments revealed that Z-100 induced IFN-beta production in these cells, resulting in induction of the 16-kDa CCAAT/enhancer binding protein (C/EBP) beta transcription factor that represses HIV-1 long terminal repeat transcription. These effects were alleviated by SB 203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), indicating that the p38 MAPK signalling pathway was involved in Z-100-induced repression of HIV-1 replication in MDMs. These findings suggest that Z-100 might be a useful immunomodulator for control of HIV-1 infection.
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PMID:Inhibition of human immunodeficiency virus type 1 replication by Z-100, an immunomodulator extracted from human-type tubercle bacilli, in macrophages. 1530 54

To enhance the in vivo antitumor activity of adoptively transferred, CD19-specific chimeric antigen receptor (CAR)-redirected cytotoxic T lymphocytes (CTLs), we studied the effect of restimulating CAR(+) CTLs through their endogenous virus-specific T-cell antigen receptor (TcR) by the cotransfer of engineered T-cell antigen-presenting cells (T-APCs). Using influenza A matrix protein 1 (MP1) as a model antigen, we show that ex vivo-expanded CD4(+) and CD8(+) T-APCs expressing a hygromycin phosphotransferase-MP1 fusion protein (HyMP1) process and present MP1 to autologous human leukocyte antigen (HLA)-restricted, MP1-specific CD4(+) and CD8(+) CTL precursors. The MP1-specific CTLs are amenable to subsequent genetic modification to express a CD19-specific CAR, designated CD19R, and acquire HLA-unrestricted reactivity toward CD19(+) leukemia and lymphoma tumor targets while maintaining HLA-restricted MP1 specificity. The restimulation of MP1xCD19 dual-specific CTLs in vivo by the adoptive transfer of irradiated HyMP1(+) T-APCs resulted in the enhanced antilymphoma potency of bispecific effector cells, as measured by elimination of the biophotonic signal of established firefly luciferase-expressing Burkitt lymphoma xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/scid) animals compared with control groups restimulated by Hy(+)MP1(neg) T-APCs. Engineered T-APCs are a novel and versatile antigen-delivery system for generating antigen-specific T cells in vitro and enhancing the in vivo effector functioning of CAR-redirected antitumor effector cells.
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PMID:Enhanced antilymphoma efficacy of CD19-redirected influenza MP1-specific CTLs by cotransfer of T cells modified to present influenza MP1. 1550 26

Entry of HIV and SIV into susceptible cells is mediated by CD4 and chemokine receptors, which act as coreceptors. To study cell entry of SIV, we constructed a cell line, xKLuSIV, derived from non-susceptible human K562 cells, that express the firefly luciferase reporter gene under control of a minimal SIV long terminal repeat (LTR). Using these susceptible cells, we studied the entry of a well-characterized molecularly cloned macrophage-tropic SIV. xKLuSIV cells that express rhesus macaque CD4 and/or the rhesus chemokine receptor CCR5 are susceptible to infection with the macrophage-tropic, neurovirulent strain SIV/17E-Fr, but only xKLuSIV cells expressing both CCR5 and CD4 were susceptible to infection by the macrophage-tropic, non-neurovirulent strain SIV/17E-Cl. CCR5-dependent, CD4-independent infection by SIV/17E-Fr was abrogated by pre-incubation of the cells with AOP-RANTES, a ligand for CCR5. In addition to viral entry occurring by a CD4-independent mechanism, neutralization of SIV/17E-Fr with rhesus mAbs from 3 different neutralization groups blocked entry into x KLuSIV cells by both CD4-dependent and -independent mechanisms. Triggering the env glycoprotein of SIV-17 EFr with soluble CD4 had no significant effect in infectivity, but triggering of the same glycoprotein of SIV/17E-Cl allowed it to enter cells in a CD4-independent fashion. Using mutant molecular clones, we studied the determinants for CD4 independence, all of which are confined to the env gene. We report here that truncation of the TM at amino acid 764 and changing a single amino acid (R751G) in the SIV envelope transmembrane protein (TM) conferred the observed CD4-independent phenotype. Our data suggest that the envelope from the neurovirulent SIV/17E-Fr interacts with CCR5 in a CD4-independent manner, and changes in the TM protein of this virus are important components that contribute to neurovirulence in SIV.
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PMID:A single amino acid change and truncated TM are sufficient for simian immunodeficiency virus to enter cells using CCR5 in a CD4-independent pathway. 1606 Dec 66

Studies on the regulation of viral transcription upon infection of the target cells have provided important information on the viral and host factors that influence pathogenesis. However, these studies have been limited so far to steady-state analysis of gene expression. Here we report an image based photon-counting method that allows real-time quantitative imaging of viral gene expression in infected single cells. Employing an HIV-1 vector bearing the firefly luciferase reporter gene, we exploited a single cell photon imaging methodology (a customized and highly sensitive imaging microscope) to measure viral gene expression following integration into a host genome in situ. Our approach reveals real-time dynamics of viral gene expression in living HIV natural target cells (primary human CD4 T cells and macrophages), and promises itself as a powerful tool for quantitative studies on a wide variety of virus-host cell interactions.
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PMID:Quantitative real-time analysis of HIV-1 gene expression dynamics in single living primary cells. 1689 17

Two new T-cell-based reporter cell lines were established to measure human immunodeficiency virus type 1 (HIV-1) infectivity. One cell line naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrates high susceptibility to HIV-1, with genomes expressing two different luciferase reporters, HIV-1 long terminal repeat-driven firefly luciferase and cytomegalovirus promoter-driven renilla luciferase. Upon HIV infection, the cells expressed firefly luciferase at levels that were highly correlated (r2=0.91 to 0.98) with the production of the capsid antigen p24. The cells also constitutively expressed renilla luciferase, which was used to monitor cell numbers and viability. The reliability of the cell lines for two in vitro applications, drug resistance phenotyping and drug screening, was confirmed. As HIV-1 efficiently replicated in these cells, they could be used for multiple-round replication assays as an alternative method to a single-cycle replication protocol. Coefficients of variation for drug susceptibility evaluated with the cell lines ranged from 17 to 41%. The new cell lines were beneficial for evaluating antiretroviral drug resistance. Firefly luciferase gave a wider dynamic range for evaluating virus infectivity, and the introduction of renilla luciferase improved assay reproducibility. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected by monitoring renilla luciferase activity.
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PMID:Use of new T-cell-based cell lines expressing two luciferase reporters for accurately evaluating susceptibility to anti-human immunodeficiency virus type 1 drugs. 1718 60

Given their self-renewing and pluripotent capabilities, human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However, the host immune response against transplanted hESCs is not well characterized. In fact, controversy remains as to whether hESCs have immune-privileged properties. To address this issue, we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death, suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses, resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover, we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally, we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together, these data suggest that hESCs are immunogenic, trigger both cellular and humoral-mediated pathways, and, as a result, are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches.
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PMID:Immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts. 1872 88

The immunological sequelae of adeno-associated virus (AAV)-mediated gene transfer in vivo is quite complex. In murine models, most AAV capsids are associated with minimal or dysfunctional T cell responses to antigenic transgene products. In this study we compared T cell activation against AAV2/8 and AAV2/rh32.33 vectors expressing nuclear-targeted LacZ (nLacZ), GFP, or firefly luciferase in murine skeletal muscle. We show that, unlike AAV8, AAVrh32.33 yields qualitatively and quantitatively robust T cell responses to both the capsid and transgene product. AAV2/rh32.33.CB.nLacZ, but not AAV2/8, drives a high degree of cellular infiltration and a loss of detectable transgene expression in C57BL/6 mice. However, cellular immunity to AAVrh32.33 is ablated in the absence of CD4, CD40L, or CD28, permitting stable beta-galactosidase expression. Treatment of CD40L(-/-) mice with the CD40 agonist, FGK45, failed to restore the CD8 response to AAV2/rh32.33.nLacZ, suggesting that additional factors are involved. Our results suggest that specific domains within the AAVrh32.33 capsid augment the adaptive response to both capsid and transgene Ags in a CD4-dependent pathway involving CD40L signaling and CD28 costimulation. Structural comparison of the AAV8 and rh32.33 capsids has identified key differences that may drive differential immunity by affecting tropism, Ag presentation or the activation of innate immunity. This murine model of AAV-mediated cytotoxicity allows us to delineate the mechanism of viral immune activation, which is relevant to the translation of AAV technology in higher order species.
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PMID:Adeno-associated virus capsid structure drives CD4-dependent CD8+ T cell response to vector encoded proteins. 1941 56

The direct detection of native proteins in heterogeneous solutions remains a challenging problem. Standard methodologies rely on a separation step to circumvent nonspecific signal generation. We hypothesized that a simple and general method for the detection of native proteins in solution could be achieved through ternary complexation, where the conditional signal generation afforded by split-protein reporters could be married to the specificity afforded by either native receptors or specific antibodies. Toward this goal, we describe a solution phase split-luciferase assay for native protein detection, where we fused fragmented halves of firefly luciferase to separate receptor fragments or single-chain antibodies, allowing for conditional luciferase complementation in the presence of several biologically significant protein targets. To demonstrate the utility of this strategy, we have developed and validated assay platforms for the vascular endothelial growth factor, the gp120 coat protein from HIV-1, and the human epidermal growth factor receptor 2 (HER2), a marker for breast cancer. The specificities of the recognition elements, CD4 and the 17b single-chain antibody, employed in the gp120 sensor allowed us to parse gp120s from different clades. Our rationally designed HER2 sensing platform was capable of discriminating between HER2 expression levels in several tumor cell lines. In addition, luminescence from reassembled luciferase was linear across a panel of cell lines with increasing HER2 expression. We envision that the proof of principle studies presented herein may allow for the potential detection of a broad range of biological analytes utilizing ternary split-protein systems.
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PMID:A general approach for receptor and antibody-targeted detection of native proteins utilizing split-luciferase reassembly. 2068 84

HIV-1 envelope glycoproteins (Envs) mediate virus entry by fusing the viral and target cell membranes, a multi-step process that represents an attractive target for inhibition. Entry inhibitors with broad-range activity against diverse isolates of HIV-1 may be extremely useful as lead compounds for the development of therapies or prophylactic microbicides. To facilitate the identification of such inhibitors, we have constructed a cell-cell fusion system capable of simultaneously monitoring inhibition efficiency and specificity. In this system, effector cells stably express a tetracycline-controlled transactivator (tTA) that enables tightly inducible expression of both HIV-1 Env and the Renilla luciferase (R-Luc) reporter protein. Target cells express the HIV-1 receptors, CD4 and CCR5, and carry the firefly luciferase (F-Luc) reporter gene under the control of a tTA-responsive promoter. Thus, Env-mediated fusion of these two cell types allows the tTA to diffuse to the target cell and activate the expression of the F-Luc protein. The efficiency with which an inhibitor blocks cell-cell fusion is measured by a decrease in the F-Luc activity, while the specificity of the inhibitor is evaluated by its effect on the R-Luc activity. The system exhibited a high dynamic range and high Z'-factor values. The assay was validated with a reference panel of inhibitors that target different steps in HIV-1 entry, yielding inhibitory concentrations comparable to published virus inhibition data. Our system is suitable for large-scale screening of chemical libraries and can also be used for detailed characterization of inhibitory and cytotoxic properties of known entry inhibitors.
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PMID:An inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of HIV-1 entry inhibitors. 2206 66

Most research on HIV transmission and microbicides focuses on the inhibition of cell-free virus (CFV) present in genital secretions. However, an effective microbicide should also block the transmission of cell-associated virus (CAV) originating from seminal T cells and macrophages. Because inhibition of CAV remains controversial, especially for viral entry inhibitors, we developed a novel in vitro assay to evaluate the activities of different classes of candidate microbicides against cell-free HIV and HIV-infected leukocytes (i.e., resting peripheral blood mononuclear cells [PBMC], activated PBMC, and monocyte-derived macrophages). The assay is based on two CD4(+) CXCR4(+) T-cell lines (R5MaRBLE and X4MaRBLE) that both contain a firefly luciferase reporter gene but differ in the expression of the CCR5 coreceptor. Consequently, the quantification of the luciferase activities and the Gag p24 concentrations in cocultures of R5-tropic HIV-infected leukocytes with each cell line separately allowed us to discriminate between the infection of the cell lines (i.e., target cells), the ongoing infection in the HIV-infected leukocytes (i.e., effector cells), and the total infection of the coculture (i.e., effector plus target cells). All 14 antiretrovirals tested were able to block target cell infection by all three sources of CAV, although a small decrease in activity (2- to 18-fold) was observed for all entry inhibitors. On the other hand, the production of Gag p24 by the infected effector cells could be blocked only by protease inhibitors. Overall, these results show that entry and protease inhibitors are eligible drug classes for inclusion in future combination microbicides.
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PMID:In vitro activities of candidate microbicides against cell-associated HIV. 2208 72


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