MXPA06005940A - Adenovirus-transfected primary cells and methods of pathway mapping - Google Patents

Abstract

A primary cell culture is transfected with a vector comprising a reporter gene operatively linked to a cis-element. Expression of a candidate regulatory protein is induced in the cell culture and its effects on the cis-element are assayed.

Description

PRIMARY CELLS TRANSFERRED WITH ADENOV1RUS AND METHODS FOR ROAD MAPPING BACKGROUND OF THE INVENTION The invention is also related to the field of path mapping and, in particular, to the pathway mapping immunocytes that are not immortal cell lines. The invention also relates to the field of modulation of the expression of intracellular signaling pathways. Description of the Related Art An intracellular signaling pathway is a series of genes whose expression is controlled by a cascade of gene-to-gene interactions triggered by an initial stimulus. In other words, the initial stimulus of an intracellular signaling pathway controls the expression of each gene on the pathway directly or indirectly through another gene of the pathway. Known signaling pathways are involved in a wide range of biological activities, including immune response, development, mitosis and inflammation and include, but are not limited to, mitogen-activated protein kinase ("MAPK") pathways, the protein kinase A ("PKA") signaling pathway, the protein kinase C ("PKC") signaling pathway, the Type I interferon signaling pathway, the calcineurin signaling pathway ("CaN") ), the cis-acting nuclear factor of the cell signaling pathway ("NF B") and the signaling pathway of the kinase L? B ("IKK".) While defects of the signaling pathways are involved In a wide range of disease states, in many cases little is known about a signaling pathway between the initial stimulus and the cellular response ultimately produced.

The mapping of the routes refers, in general, to the elucidation of the steps between the initial stimulus of a road and the final answers. In the context of the present invention, "path mapping" refers to the determination of whether a regulatory element of action in cis in power, is controlled, either directly or indirectly, by a given signaling path. A known method of route mapping employs host cells from a cultured cell line that are transfected with a DNA plasmid containing a reporter gene operably linked to a regulatory element to be evaluated. The use of reporter genes is well recognized in the art. Kain, SR, and Ganguly, S, Overview of Genetic Reporting Systems, Unit 9.6 in Current Protocols in Molecular Biology, Ed. Ausubel, FM, et al., (Wiley and Sons, NY, 1995). Transfection refers to the introduction of foreign DNA into a host cell, such as a cultured mammalian cell. Transfection has shown SRE a powerful tool to analyze the regulation of functions and the interaction of genes and their respective gene products. Methods for transfection of cells are well recognized in the art. The methods of chemical and physical transfection include DEAE dextran (Fox RM, et al, Biochemistry Oct 4; 16 (20): 4470-7, PMID 911769 (1977)), calcium phosphate (Pear, WS, Nolan, GP, Scott, M.L, &Baltimore, D .. Proc. Nati, Acad. Sci. USA 90, 8392 (1993)), liposome-mediated transfection (Zhang WW, et al., Biotechniques Nov; 15 (5 ): 868-72 (1993)), microinjection (Colbere-Garapin F, and Garapin AC, Dev Biol Stand; 55: 267-71, PMID: 6329857 (1983)), and electroporation (Neumann E and Kakorin S, Technol Cancer Res Treat, Oct; 1 (5): 329-40 (2002)). The use of recombinant adenovirus vectors for transfection of the membrane is ~ ñTuy recognized in the art. "Berkner, KL and Sharp, PA Nucelic Acids Res. 11: 6003-6020 (1983); Berkner, KL and Sharp, PA BioTechniques 6: 616-629 (1988); Bett, AJ, Haddara, W, Prevec, L and Graham, FL PNAS USA 91: 8802-8806 (1994), Hitt, M, Addison, CL, and Graham, FL Adv. Pharmacol. 40: 137-206 (1997). Recombinant adenoviruses have been used to transfect, inter alia , cardiac tissue, Ardehali, AJ Thor, Cardiovas, Surg 111: 246-252 (1996) and lymphocytes, Leon, RP, Hedlund, T, Meech, SJ, Li, S, Schaack, J, Hunger, SP, Duke, RC, and DeGregori, J PNAS USA 95: 13159-13164 (1998). Human adenoviruses are double-stranded DNA viruses that enter cells by receptor-mediated endocytosis. These viruses are considered very suitable for gene transfer because they are easy to grow and manipulate and exhibit a wide range of hosts both in vivo and in vitro. Adenoviruses can infect inactive and replicating target cells and persist extrachromosomally, instead of integrating into the host genome. Recombinant adenoviruses can give rise to relatively large segments of foreign DNA (approximately 7 kb) and have the advantage of high titer viral production. Among the known vectors for reporter constructions used for route mapping is the plasmid pCREd21EGFP, marketed by BD Biosciences Clontech. This plasmid contains a cyclic AMP response element ("CRE") operably linked to a green fluorescent protein ("GRF") reporter gene and can be used to measure activity in pathways such as the N kinase signaling pathway. -terminal Jun ("JNK"), the p38 MAPK signaling pathway and the signaling pathway of protein kinase A ("PKA"). This plasmid uses the pUC origin of replication for the propagation of E. coli and a viral f1 origin for the production of a single-stranded DNA. This plasmid is transfected into eukaryotic cells with much lower efficiency than the adenovirus-based vectors and are only efficient enough to use in cultured eukaryotic cell lines and not in primary eukaryotic cell cultures. Since known vectors for immunocyte pathway mapping are not efficient enough to be practical for use in primary immunocytes, path mapping using the methods of the prior art can only be carried out at present in cultured cell lines. . However, immortalized cell lines tend to have protein expression patterns significantly different from those of their non-immortalized counterparts. It is convenient to be able to map the pathways in primary host cells of higher organisms without unnecessarily breaking the normal pattern of cell expression. It is especially advantageous to be able to map the pathways in primary immunocytes without having to transform them into immortal cell lines. BRIEF SUMMARY OF THE INVENTION In one aspect, the invention relates to a method for determining whether a stimulus, which is known to modulate the expression of a signaling pathway in an immunocyte, has the ability to modulate the expression of an element in cis in potency, which includes the steps of transfecting into the immunocyte a recombinant adenovirus having a reporter gene operably linked to the element in potential cis; measure a base level of reporter gene activity; apply the stimulus to the immunocyte and measure the activity of the reporter gene in response to said stimulus. In another aspect, the stimulus consists of modulating the expression of a regulatory protein. In another aspect, the method further comprises co-transfection with an expression system for the regulatory protein. In an alternative aspect, the stimulus introduces a potential regulatory compound. In another aspect the reporter gene is the luciferase gene, green fluorescent protein ("FGP"), β-galactosidase ("GAL"), chloramphenicol acetyltransferase ("CAT") or a suppressor gene such as Bsd. In another aspect, the element in cis is related to inflammation. In another aspect, the cis-element is AP-1, CRE, ISRE, NFAT, NFKB, or SRE. In another aspect, the immunocyte is a macrophage, a CD4 + T cell or an immature dendritic cell. In one aspect, the invention relates to a method for inhibiting the expression of a signaling pathway in an immunocyte, which comprises the steps of transfecting the immunocyte with a recombinant adenovirus containing a suppressor gene operably linked to a belonging cis-element. to the signaling pathway and induce the expression of the suppressor gene. In a further aspect, the signaling path is the NFKB signaling path. In another aspect, the suppressor gene is l? Bsd. In another aspect, the immunocyte is a macrophage, a CD4 + T cell or an immature dendritic cell. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A illustrates macrophages transfected with a recombinant adenovirus capable of expressing the green fluorescent protein ("AdV: GFP"). Figure 1B illustrates dendritic cells transfected with a recombinant adenovirus with the ability to express the green fluorescent protein ("AdV: GFP"). Figure 1C illustrates the expression of green fluorescent protein in lymphocytes T. Figure 1 D illustrates the expression of green fluorescent protein in B lymphocytes. Figure 2 illustrates two different cell cultures, one transfected with a recombinant adenovirus capable of expressing a superdominant mutant of L? B ("Adv: l? Bsd ") and the other transfected with a recombinant adenovirus capable of expressing the green fluorescent protein (" AdV: GFP "), each one illustrated with contrast and staining with propidium iodide. Figure 3A is a schematic representation illustrating a superdominant mutant of IKB ("lβ Bsd") that blocks the NFKB signaling pathway. Figure 3B is a graph illustrating the effects of the inhibition of NFKB activation on the expression of inflammatory cytokines during the maturation of dendritic cells. Figure 4 is a schematic representation illustrating a promoter / reporter system based on recombinant adenovirus. Figure 5A is a graph illustrating the activation of cis CRE elements in 00 * 7 cells upon stimulation. Figure 5B is a graph illustrating the activation of cis AP-1 elements in CD4 + T cells upon stimulation. Figure 5C is a graph illustrating the activation of cis elements NFKB in CD4 + T cells, before stimulation. Figure 5D is a graph illustrating the activation of cis ISRE elements in CD4 + T cells upon stimulation. Figure 6A is a graph illustrating the activation of cis elements in macrophages. Figure 6B is a graph illustrating the activation of cis elements in dendritic cells. Figure 7A is a schematic representation of the results of a hypothetical path mapping experiment. Figure 7B is a schematic representation of the results of a hypothetical path mapping experiment.

DETAILED DESCRIPTION OF THE INVENTION The present invention presents methods and compositions for determining the pathway of regulatory elements that act in cis in the signaling pathways of primary immunocytes. In the context of the present invention, the term "cis-acting regulatory element" is used interchangeably with "element in cis" and refers to a polynucleotide regulatory element 1 operatively linked to a coding sequence, wherein the regulatory element modulates the expression of the coding sequence. In the context of the present invention, the expression "intracellular signaling pathway" is used interchangeably with "signal transduction pathway", "signaling pathway" and "pathway" and refers to a collection of genes whose regulation is modulated, either directly or indirectly, by a given initial stimulus. In a typical signaling pathway, the initial stimulus activates a first element in cis that activates the production of a corresponding first regulatory protein. Next, the first regulatory protein activates a second cis-element of the pathway, which in turn activates the production of a second regulatory protein. The second regulatory protein then activates a third element in cis, and so on. In this example, the initial stimulus regulates the third element in cis indirectly. In the context of the present invention, an element in cis is identified as belonging to the pathway if it is regulated directly or indirectly by the initial stimulus of the pathway. In the context of the present invention, "initial stimulus" refers to any phenomenon that modulates the activity of a pathway. Initial stimuli include, but are not limited to, increased or reduced expression of a regulatory protein, viral infection, contact with an allergen, DNA damage and extracellular stress. Examples of extracellular stress include, but are not limited to, thermal shock, osmotic stress, osmotic stress, pH tension, and ionizing radiation. Many human diseases originate in one or more defects of the signaling pathway of immune cells such as macrophages, T cells and dendritic cells. For example, defects of the p38 mitogen-activated protein kinase pathway ("MAPK") have been causally implicated, inter alia, in arthritis, sepsis and infection by the human immunodeficiency virus. Likewise, defects in the signaling pathway of type I interferon give rise to an insufficient response to viral infection. The following describes some known signaling pathways and their component cis elements. Regulatory elements of action in cis and its pathways A regulatory element of action in cis, also known as an element in cis, is a polynucleotide regulatory element operatively linked to a coding sequence, where the regulatory element modulates the expression of the coding sequence. Examples of cis-acting regulatory elements include, but are not limited to, the activating protein a ("AP-1"), the cyclic AMP response element ("CRE"), the element to the response to stimulation with interferon ("ISRE"), nuclear factor of activated T cells ("NFAT"), nuclear factor of KB cells ("NFKB") and serum response element ("SRE"). The enhancer element of cis-activating protein 1 ("AP-1") is modulated by the transcription factors c-jun and c-fos. The enhancing element of cis-acting AP-1 appears in the signaling pathway of the N-terminal c-jun protein kinase ("JNK"). The JNK signaling pathway is a class of mitogen-activated protein kinase ("MAPK") pathway and the MAPK signaling pathway has been implicated in the cellular response to various forms of extracellular stress, as well as cellular sensitivity to apoptosis The enhancer element of the cis-action cyclic AMP response ("CRE") is modulated by the transcription factors AT2 and CREB. The enhancing element of CRE of action in cis CRE appears in the JNK signaling pathway, the p38 signaling pathway and the signaling pathway of Protein Kinase A ("PKA"). As with the JNK signaling pathway, the p38 signaling pathway is a class of mitogen-activated protein kinase pathway ("MAPK") and has been implicated in a cellular response to various forms of extracellular stress and inflammatory cytokines . The signaling pathway of PKA has been implicated in mitosis. The enhancer element of the response element to stimulation with cis-acting interferon ("ISRE") is modulated by the transcription factors STAT1 and STAT2. The cis-acting ISRE enhancer element appears in the signaling pathway of Type I interferon. The signaling pathway of Type I interferon in the cellular response to viral infections has been implicated. The nuclear factor enhancing element of activated cis-acting T cells ("NFAT") is modulated by the transcription factor NFAT. The cis-acting NFAT enhancer element appears in the signaling pathway of protein kinase C ("PKC") as well as in the signaling pathway of calcineurin ("CaN"). The signaling pathway of PKC has been implicated in the action of histamine on vasoconstriction. The CaN signaling pathway has been implicated in cardiac hypertrophy. The nuclear factor-enhancing element of cis-acting KB cells ("NFKB") is modulated by the transcription factor NFKB. The enhancing element of cis-acting NFKB appears in the signaling pathway of kinase lβB. The signaling pathways of IKK and NFKB have been implicated in the cellular response to various forms of extracellular stress. The enhancer element of the cis response element ("SRE") is modulated by the transcription factors Elk-1 and SRF. The enhancing element of the cis-acting SRE appears in the MAPK signaling pathway as well as in the JNK signaling pathway. It is known that the aforementioned elements in cis correspond to the respective ways listed above. Nevertheless, there are numerous elements in cis whose membership in the signaling routes has not yet been determined. An embodiment of the present invention presents a method for determining whether an element in cis in power belongs to a given pathway. In this embodiment, a reporter construct containing the candidate cis-element operably linked to a reporter gene in a primary host cell such as an immunocyte is first transfected. Then the host cell is stimulated so that it activates the pathway in question. The activity of the reporter gene is then measured. If the activity of the reporter gene is modulated in response to the stimulus, then it is determined that the candidate cis element belongs to the pathway in question. Construction and Administration of the Reporting Construction One embodiment of the invention uses a recombinant reporter construct comprising a reporter gene operatively linked to a candidate cis element. In one embodiment, this recombinant reporter construct is administered to a host cell by means of an adenovirus-based vector. Figure 4 is a schematic representation illustrating a recombinant vector based on adenovirus. In a reylation, this vector is derived from the genome of an adenovirus of type 5, such as the BD ADENO-X DNA, which has a length of 33 kb and which has been modified by deletion of large portions of the E1 and E3 regions. . The insertion of the recombinant reporter comprises the element in cis candidate, a transcription blocker before the element in cis candidate and a reporter gene after the element in cis candidate. The blocking element of the previous transcript ("TB") reduces the levels of background transcription of the reporter gene and may include one or more pause elements of the transcription or polyadenylation sites. Typical reporter genes include, but are not limited to, luciferase, β-galactosidase ("GAL"), chloramphenicol acetyltransferase ("CAT"), green fluorescent protein ("GFP") and variants thereof, including, although not limited to destabilized variants. In one embodiment, which is described below in more detail, the reporter gene is a superdominant mutant with the ability to block the expression of a signaling pathway. In one embodiment, the recombinant reporter insert is flanked by a l-Ceu I restriction site and a Pl-Sce I restriction site and ligated in vitro to linearized ADENO-X DNA, which is previously cut with l-Ceu I and Pl-Sce I. The DNA thus obtained is grown in E. coli and the purified plasmid is transfected into HEK293 cells. The recombinant viruses are rescued by transfecting the recombinant adenovirus DNA in its entirety in HEK293 cells and then amplified to generate base materials with high titer. Determination of whether an Element in cis Candidate Belongs to a Pathway Once the reporter construct containing the element in cis candidate is cloned into the adenovirus-based vector, the reporter construct is then transfected into a host cell by putting the vector in contact with the host cell in a multiplicity of sufficient infection ("MOI") and incubating for a sufficient period of time. MOIs and appropriate incubation times are presented in the following examples. Once it has been transfected into the host cell with the reporter construct containing the candidate cis element, the base level of activity of the reporter gene is measured. Then a stimulus known to activate the pathway in question is applied to the cell. For example, if the pathway in question is the p38 MAPK pathway, the stimulus applied could SRE an inflammatory cytokine, or cellular stress such as heat shock or a hypertonic medium. After applying the stimulus, the activity of the reporter gene is measured and compared with its level of basal activity. If the activity of the reporter is modulated by the application of the stimulus, then it is determined that the candidate cis element belongs to the path in question. EXAMPLES The following examples are non-limiting. For example, the use of cis elements other than those listed is contemplated, as well as the use of other reporter and regulatory genes that are not mentioned either. In the context of the present invention, the term "ex vivo" refers to the use of primary cell cultures, unlike the use of immortalized cell lines. Example: Recombinant Adenovirus Infection in Human Primary Immunocytes In this example, all major types of human primary immunocyte cells are examined for their susceptibility to adenoviral infection using a recombinant adenovirus capable of constitutively expressing green fluorescent protein ( "Adv.GFP"). Said recombinant adenovirus can be purchased from Quantum Biology. In this example, the types of human primary immune cells successfully transfected by AdV: GFP include bone marrow macrophages, mature dendritic cells differentiated in vitro ("kDCs") and peripheral blood mononuclear cells ("PBMCs"). The iDCs are prepared from negatively selected CD14 + cells of the PBMCs by IL-4 and GM-CSF. The macrophages and iDCs are infected with AdV: GFP in a multiplicity of infection in the range of 10 to 330 per cell, then incubated for 16-24 hours. The cells are then examined under a fluorescent microscope to determine GFP expression. Figure 1A illustrates the expression of GFP in transfected bone marrow macrophages and Figure 1B illustrates the expression of GFP in iDCs at the rate of 100 MOI. At this level of MOI, up to 90% of the cells exhibit detectable GFP expression. Fresh peripheral blood mononuclear cells ("PBMCs) are infected with AdV: GFP at an MOI in the range of 100 to 1000 per cell." Twenty-four hours after infection, infected cells are labeled with monoclonal antibodies specific for CD4 and CD19. Figure 1C illustrates the expression of GFP in CD4 + T lymphocytes and Figure 1D illustrates the expression of GFP in CD19"B lymphocytes infected by AdV: GFP in an MOI of 300. In both figures, the expression of GFP with FL1 adjusted to 414 nm using a FACSCalibur analyzer, marketed by BD Biosciences. The fluorescent intensity change illustrated in both cited figures represents a majority of cells in both cell cultures that exhibit detectable GFP expression. Example: Blockade of the NFKB Signaling Pathway in Macrophages with AdV: l? Bsd In this example, an adenovirus capable of constitutively expressing a superdominant L? B mutant ("AdV: l? Bsd") is constructed. The kBsd mutant comprises two point mutations in human l? Ba which changes serine 32 and serine 36 to alanines. Accordingly, the mutant L? Bsd can not be phosphorylated by S? B qunase-b. In this way, the kBsd mutant can block the NFKB signaling pathway by preventing degradation mediated by the proteasome of the L? Ba. In the absence of the NFKB signaling pathway, such as when blocked by the superdominant mutant L? B, treatment with TNF-a induces apoptosis in various cell types, including macrophages. In this example, bone marrow macrophages are infected with AdV: GFP as control or with AdV: l? Bsd. Next, both series of macrophages are treated with TNF-a at a rate of 10 nM. Apoptosis is measured by staining with propidium iodide ("pi") that stains DNA in the absence of permeabilization of cells. As illustrated in Figure 2, the AdV: GFP control group - whose NFKB signaling pathway remained intact - exhibited minimal induction of apoptosis, of less than 5% of the total cells. (The uninfected control and the infected cells are treated with TNF-a also exhibited less than 5% induction of apoptosis). Conversely, cells treated with AdV: l? Bsd exhibited induction of apoptosis in more than 75% of the cells. Accordingly, it is shown that AdV: l? Bsd effectively blocks the NFKB signaling pathway in macrophages. Example: Blockade of the Signaling Pathway of NFKB in Dendritic Cells with AdV: l? Bsd In this example, the effect of the blocking of the Pathway of NFKB signaling in in vitro differentiated human dendritic cells ("iDCs") Figure 3 is a schematic diagram of the signaling pathway of the iDCs after activation with lipopoplisaccharides ("LS") or by CD40 ligand ("CD40L"), with blockade of the NFKB signaling pathway by l Bsd LPS activates TLR4, CD40L activates CD40 In this example, iDCs are obtained in the following manner: First, peripheral blood mononuclear cells are obtained (" PBMCs ") by centrifugation at a density gradient A centrifugation medium at a density gradient is FICOLL PAQUE, marketed by Amersham Biosciences, then monocytes are isolated from the PBMCs by magnetic depletion of the non-monocytes. the iDCs of the monocytes by treating the monocytes with IL4 and GM-GSF for 6 days In this example, a simulated infection is subsequently (1), (2) the resting cultures of iDC are infected s with AdV: GFP at an MOI of 100 as a control or (3) is infected with AdV: l? Bsd at an MOI of 100. (In the context of the present invention, "multiplicity of infection" ("MOI" ) refers to the ratio of infectious units to infected cells. Unless otherwise indicated, the multiplicity of infection is expressed in terms of infectious units to T cells HEK293). After an infection period of 16-24 hours, then (1) each series of iDCs is activated in a simulated manner, (2) it is activated by LPS at a concentration of 1 μg / ml or (3) it is activated by CD40L in a concentration of 1 μg / ml. The determination of the effects of inhibition of NFKB activation on the expression of inflammatory cytokines during the maturation of iDCs was carried out by real-time PCR analysis of selective cytokines and chemokines, as well as a cytometric bead test to determine the secretion of various cytokines. At 4-8 hours after activation, the RNA is extracted and the levels of expression of various cytokines and chemokines are measured by TaqMan PCR in real time. In addition, supernatants are collected at 8 hours after activation and examined for cytokine secretion by cytometric bead assay. Figure 3B is a graph illustrating the expression of selected cytokines in AdV: lβ Bsd infected cells compared to cells infected with AdV: GFP. In this example, it is demonstrated that the AdV: l? Bsd effectively blocks the NFKB signaling pathway in activated dendritic cells. Specifically, a considerable reduction in the expression of 1L-12, IL-10, IL-8 and IL-6 is demonstrated in this figure. Example: Comparison of AdV: l? Bsd with S0101627 In this example, an exhaustive study of the inhibition of the NFKB signaling pathway in dendritic cells by LPS or CD40 ligand is carried out using a parallel comparison of the inhibition of expression of L? B superdominant or by a known inhibitory compound of the beta L-B kinase, S0101627. In this example, (1) the CDI infection is simulated, (2) it is infected with AdV: GFP as a control or (3) it is infected with AdV: l? Bsd for 12 hours, and then stimulates with LPS or CD40L. The CDIs not infected with S0101627 are treated at the time of stimulation. 8 hours after the stimulation, the supernatants and the cells are collected and reprocessed by the analysis of cytometric beads to determine the inflammatory cytokines and by real-time PCR for the expression of selective cytokines / chemokines, respectively. Tables 1 and 2 show the PCR data in real time with respect to the expression of cytokines and chemokines, respectively.

Table 1. Expression of selective cytokines when inhibiting the signaling pathway of NFKB by means of a chemical inhibitor (comp) or by biochemical means using the adenovirus (AdV: l? B) Control Dev. Std Comp Dev. Std AdV: GFP Dev. StdAdV: lkB Dev. Std IL-1ß cont 29,263 3,529 34,064 2,985 14,642 0,112 13,813 2,783 LPS 4602,917 116,731 185,004 15,267 10032,276 274,488 241,921 21,914 CD40L 12262,184 269,214 422,791 10,747 16591, 932 1384,342 422,584 18,477 IL-6 cont 6,931 0,549 9,073 1, 482 60,976 1,680 18,473 2,771 LPS 1,448,169 110,883 10,393 5,527 4805,537 97,018 104,716 5,681 CD40L 1538,403 55,857 4,601 1, 040 6710,969 224,149 136,214 4,596 IL-8 cont 417,177 38,844 7008,142 306,451 895,411 28,116 949,533 15,990 LPS 33310,859 669,480 29,927,240 1011, 946 59572,640 1490,781 6117,183 314,083 CD40L 52413,686 710,084 26603,448 466,182 84912,576 832,407 9403,936 252,355 IL-10 cont 58,132 9,059 23,929 2,189 122,912 3,738 69,419 2,771 LPS 131, 546 10,705 27,550 5,941 1317,157 6,710 113,991 12,935 CD40L 503,517 13,998 14,016 1,442 3213,362 29,764 210,082 2,872 IL-12 cont 0.489 0.159 0.035 0.000 0.166 0.299 0.067 0.000 LPS 367.059 26.466 0.321 0.056 3275.257 113.158 33.137 6.168 CD40L 3969.080 236.063 0.270 0.096 8956.589 512.201 239.002 14.327 TNF-a cont 96,467 2,508 277,594 2,824 327,274 7,669 105,575 8,647 LPS 2085,233 58,702 508,459 12,790 6372,957 5,395 1345,425 65,059 CD40L 892,387 6,614 244,186 13,417 7137,164 155,537 2802,769 151, 089 Table 2. Expression of selective chemokines upon inhibition of the NFKB signaling pathway by means of a chemical inhibitor (comp) or by biochemical means using the adenovirus (AdV: l? B) Dev. Desv. Desv. Control Std comp Std AdV: GFP DßSV. Std AdV: lkß Std CCL4 Cont, 78.24 9.65 391.81 33.49 344.23 15.58 81.04 4.70 LPS 5506.79 207.13 731.64 19.09 16431.08 545.57 1052.61 115 , 21 CD40L 2052.99 111.58 795.14 83.12 11834.94 136.51 3497.32 12.78 EXODUS-1 Cont, 0.05 0.00 0.00 0.00 0.12 0.01 0.20 0.00 LPS 21, 33 5.07 0.23 0.00 64.09 4.58 1, 15 0.39 CD40L 98.24 21, 26 0.21 0.04 185.96 30.25 10.30 1.05 GRO-1 Cont, 0.59 0.29 2.75 0.98 1.87 0.12 0.98 0.20 LPS 106.76 23.26 5.77 0.50 296.28 28.22 7, 88 4.48 CD40L 225.25 44.53 3.68 1.43 353.45 75.34 19.20 1.88 MIG Cont, 0.45 0.19 0.38 0.21 17.79 1, 57 0.55 0.33 LPS 447.49 37.46 0.76 0.42 925.99 36.47 20.45 2 , 49 CD40L 3.58 0.37 0.27 0.19 911, 60 87.27 9.50 1.50 SCYA3 Cont, 88.52 8.01 284.19 18.69 417.14 77.22 167.58 10.86 LPS 2578.07 358.01 1590.39 406.06 6361.56 681.21 1297.35 157 , 74 CD40L 1055.66 '74.36 1311.47 258.38 6604.14 1174.11 2853.10 94.21 TARC Cont, 655.20 90.76 502.24 236.85 718.52 194.34 631, 01 81, 71 LPS 1602.37 343.61 678.33 104.44 1337.75 140.59 602.76 63 , 24 CD40L 4303.55 637.38 440.39 70.93 1448.82 225.74 624.01 63.94 SCYA19 Cont, 0.32 0.13 0.46 0.35 2.76 0.51 0.72 0.39 LPS 298.64 11.87 0.21 0.08 1207.98 74.16 2.22 0 , 34 CD40L 119.31 6.81 0.27 0.15 1682.39 44.62 2.22 0.22 RANTES Cont, 14.49 2.70 13.89 1, 29 401, 75 57.96 18.88 1.97 LPS 7512.60 4992.80 18.05 4.18. 16745.39 2834.61 279.75 18.42 CD40L 1274.12 283.33 16.41 1.56 14526.19 5679.46 75.28 7.54 SCYB11 Cont, 0.66 0.00 0.33 0.00 1029.52 76.69 1.08 0.46 LPS 2343.81 382.47 1.30 0.23 8467.22 1551.04 224.25 52 , 98 1 £) CD40L 11.73 2.70 0.45 0.29 5928.08 737.52 86.70 11.39 SCY1 Cont, 0.32 0.24 0.12 0.12 0.60 0.11 0.18 0.00 LPS 132.21 33.22 0.63 0.34 164.18 11.05 24.59 2 , 33 CD40L 28.39 1.80 0.39 0.24 51.26 3.57 22.25 5.19 SCYA10 Cont, 4,56 1,57 5,40 0,56 10,16 2,55 3,55 0,94 LPS 7,72 1,34 6,49 0,14 8,43 2,69 5,29 1 , 15 CD40L 1, 81 0.15 1, 95 0.42 12.10 0.92 3.43 1.51 SCYA22 Cont, 942.96 65.13 518.31 12.32 861.27 34.95 974.96 26.85 LPS 3362.80 116.46 658.42 33.05 5233.39 280.99 1169.51 82 , 86 CD40L 7862.22 135.85 527.75 42.69 8413.07 261, 49 886.01 59.45 GR02 Cont, 0.83 0.41 10.70 7.99 6.22 0.97 1.62 0.97 LPS 48.08 4.84 24.01 3.40 88.46 69.96 12.96 0 , 58 CD40L 77.61 13.39 37.00 8.75 126.88 12.70 10.71 4.50 These results demonstrate a high efficiency of transfection, as well as the inhibition of the NFKB signaling pathway comparable with the of compound S0101627. Example: Generation of a Recombinant Adenovirus with Ability to Express a Reporter or Regulator Gene Under the Control of an Inflammation-Linked Selective Element in cis This Example illustrates the construction of recombinant adenovirus constructs to map the signaling pathways related to inflammation. Subsequent examples describe its use. In this example, the recombinant adenoviruses are constructed on the basis of the BD ADENO-X polynucleotide, marketed by BD Biosciences Clontech. The use of other adenoviruses is contemplated and the use of ADENO-X should not be considered as limiting. The ADENO-X viral DNA is approximately 33 kb in length. It is obtained from the genome of an adenovirus type 5, and has been altered by deletion of large portions of the E1 and E3 region of the adenovirus. Figure 4 presents a schematic diagram of the recombinant adenovirus vector and the promoter / reporter system inserted therein. As illustrated in Figure 4, the insert contains a transcription blocker prior to a cis-acting enhancer element that is operably linked to a luciferase reporter gene. The six elements in cis illustrated in Figure 4 are examples and are not limiting. The possibility of using other elements in cis in addition to those listed is contemplated. The use of other reporter genes is contemplated instead of that of luciferase. It is also contemplated that the insertion may contain a regulatory gene such as the superdominant mutant l? Bsd in place of a reporter gene. As illustrated in Figure 4, cloning of the insert in the E1 region of the recombinant adenovirus is carried out. This recombinant adenovirus construct is rescued by transfecting it into HEK293 cells, which are then amplified to produce adenovirus base materials with high titer. A viral titer of approximately 1x10 9 pfu / ml is usually obtained in HEK293 after two amplification cycles. Example: Infection of Human Primary Immune Cells by Recombinant Adenovirus Reporters In this example, recombinant adenoviruses capable of expressing a reporter gene under the control of cis-specific elements are used to map specific signaling pathways. Table 3 lists cis-related elements related to inflammation that are used to generate these recombinant adenoviruses, together with their respective transcription factors and signaling pathways.

Table 3. Signaling Pathways Related to Inflammation In this example, the reporter recombinant adenoviruses of the previous example are used to infect primary human immune cells. The infected cells are then stimulated and the reporter activity is observed in response to the stimulation. The stimulation applied to infected cells depends on the type of infected cell. For example, CD4 + cells are stimulated with PMA / lonomycin ("Pl") or antibodies against CD3 and CD28 in the presence of the G protein. On the other hand, macrophages such as macrophages adhered to plates derived from monocytes CD14 + are stimulated with lipopolysaccharide ("LPS"). In the same way, dendritic cells such as dendritic cells differentiated in vitro with LPS are stimulated. After a certain period after the stimulation, the cells are collected and the luciferase activity is measured. The luciferase activity can be measured by means of an enzymatic assay using a photon-generating substrate and detecting the photons by means of a luminometer. In this example, CD4 + T cells are prepared in the following manner: First, whole blood mononuclear cells ("PBMCs") are obtained by centrifugation in a density gradient. A means of centrifugation at a density gradient is FICOLL PAQUE, marketed by Amersham Biosciences. The CD4 + T cells of the PBMCs are then isolated by magnetic depletion of the CD8 + T cells, B cells, NK cells and monocytes. In this example, resting cultures of CD4 + T cells are then infected with a recombinant adenovirus containing the luciferase gene that is transcribed under the control of a specific cis-element. In this example four constructs of recombinant adenoviruses are used to infect four separate cultures of resting CD4 + T cells, each of which contains one of four cis-elements: CRE, AP-1, NFKB and ISRE. After 16 hours of incubation after infection, (1) a simulated stimulation of the cells ("C") is performed, (2) they are stimulated with 20 mg / ml of PMA and 0.5 ug / ml of lonomycin ("Pl") or (3) are stimulated with 10 ng / mg anti-CD3 mAb, 5 ug / ml anti-CD28 mAb and 10 ug / ml Protein G ("3-28"). At 8 hours, 24 hours and 48 hours after the stimulation, the cells are harvested and the luciferase activity is measured. The luciferase activity can be measured by means of a luminometer. Figure 5A is a graph illustrating the luciferase activity of CD4 + T cells infected with a recombinant adenovirus containing an element in cis CRE. Figure 5B is a graph illustrating the luciferase activity of CD4 + T cells infected with a recombinant adenovirus containing an element in cis AP-1. Figure 5C is a graph illustrating the luciferase activity of CD4 + T cells infected with a recombinant adenovirus containing an element in cis NFKB. Figure 5D is a graph illustrating the luciferase activity of CD4 + T cells infected with a recombinant adenovirus containing an element in cis ISRE. These figures exhibit considerable activation of the elements in cis CRE and AP-1 48 hours after stimulation with Pl and no consideration of any other combination of cis-element and stimulation. Similar experiments were performed to detect macrophages bound to the CD14 + plates. In this example, macrophages are obtained in the following manner: First, mononuclear cells and whole blood ("PBMCs") are obtained by centrifugation in a density gradient. A means of centrifugation at a density gradient is FICOLL PAQUE, marketed by Amersham Biosciences. Then monocytes are isolated from PBMCs by magnetic depletion of non-monocytes. Finally, monocyte macrophages are isolated by adhesion of the monocytes to a tissue culture plate in the absence of fetal bovine serum ("FBS"). In this example, it is then infected to the resting cultures of isolated macrophages with a recombinant adenovirus containing the luciferase gene which is transcribed under the control of a specific cis-element. In this example, recombinant adenovirus constructs are used to infect four separate macrophage cultures, where each construct contains one of four cis-elements: CRE, AP-1, NFKB and ISRE. Macrophages are infected with an MOI of 100 per cell. After 16 hours of incubation after infection, the stimulation of the macrophages ("control") is simulated or stimulated with lipopolysaccharide ("LPS") at a concentration of 1 μg / ml. 24 hours after the stimulation, the cells are harvested and the luciferase activity is measured by means of a luminometer. Figure 6A is a graph illustrating the luciferase activity of four groups of macrophages, each of which has been infected with one of four recombinant adenoviruses, where each recombinant adenovirus contains an element in cis AP-1, a cis-element. CRE, an element in cis ISRE and an element NFKB, respectively. This figure demonstrates a considerable activation of the base level, as well as a significant stimulated activation of the elements in cis AP-1 and CRE. Similar experiments were carried out for dendritic cells differentiated in vitro ("iDCs"). In this example, the iDCs are obtained in the following manner: First, peripheral blood mononuclear cells ("PBMCs") are obtained by centrifugation at a density gradient. A means of centrifugation at a density gradient is FICOLL PAQUE, marketed by Amersham Biosciences. Monocytes are then isolated from the PBMCs by magnetic depletion of the monocytes. Finally, the iDCs of the monocytes are isolated by treating the monocytes with IL4 and GM-GSF for 6 days. In this example, the resting cultures of iDCs are subsequently infected with an adenovirus containing the luciferase gene that is transcribed under the control of a specific cis-element. In this example, three constructs of recombinant adenoviruses are used to infect three separate cultures of iDCs, where each construct contains one of three cis-elements: AP-1, NFKB and ISRE. The iDCs are infected with an MOI of 100 per cell. After 16 hours of post-infection incubation, the stimulation of the iDCs ("C") is simulated or stimulated with lipopolysaccharide ("LPS") at a concentration of 1 μg / ml. After 2 hours 4 hours, 8 hours and 24 hours of the stimulation, the cells are harvested and the luciferase activity is measured. Figure 6B contains three graphs illustrating the time course of stimulation with AP-1, NFKB and ISRE, respectively. These graphs show significant stimulation by the three elements in cis. These graphs also indicate different courses of activation over time. Example: Use of the Adenovirus Reporter System for the objective Validation and the habilitation of in vitro compounds in the Drug Decontamination process. The above examples demonstrate that the selective activation, as well as the activation over time of transcription factors in human primary immune cells after specific stimulation. These observations allow the use of these reporter systems of activation of adenovirus-based transcription factors both for objective testing and for enabling compounds in the drug discovery process. Figures 7A and 7B are schematic representations illustrating this process. In this example, the target cells are infected with different reporters of recombinant adenoviruses, which appear as columns 1 to 6 in Figures 7A and 7B. Then stimuli are added to the crops. After incubation for a certain period of time, the luciferase activities are measured. The full circles represent hypothetical luciferase activity. The lack of luciferase activity or the one that is not detectable without treatment with a stimulus would not be registered. If a stimulus activates the binding of the transcription factor to its response element, for example, c-jun binds to an AP-1 enhancing element, transcription would be induced and luciferase activity could be detected. Different stimuli, represented by rows A and B of Figure 7A, can activate different signaling pathways. Once this is established, co-infection of dominant negative mutants such as lβ Bsd, of a target gene, in combination with a reporter adenovirus can be obtained. Activation of the specific signaling pathway in which the target is potentially involved can be reduced (and recorded as reduced luciferase activity) in its activation upon specific stimulation. Figure 7B is a schematic representation illustrating the activity of the recombinant adenovirus reporter in the presence and absence of coinfection of a dominant negative mutant.

Claims (14)

1. CLAIMS Having thus specially described and determined the nature of the present invention and the manner in which it has been carried out in practice, it is claimed to claim as property and exclusive right. 1. A method to determine if a stimulus has the capacity to activate a regulatory element of action in cis in an immunocyte, where said regulatory element of action in cis is regulated by at least one transcription factor or enhancer and where it is known that said stimulus modulates the expression of a signaling pathway, which method comprises the following steps: (a) transfecting into the immunocyte a recombinant adenovirus, a recombinant adenovirus comprising a reporter gene operatively linked to said cis-acting regulatory element; (b) measuring a base level of activity of the reporter gene; (c) applying said stimulus to said immunocyte and (d) measuring the activity of the reporter gene in response to said stimulus.
2. 2. The method according to claim 1, wherein said stimulus comprises modulating the expression of a regulatory protein and said application step (c) comprises modulating the expression of said regulatory protein.
3. 3. The method according to claim 2, further comprising the step of co-transfecting said immunocyte with an expression system for said regulatory protein.
4. 4. The method according to claim 1, wherein said application step (c) comprises introducing a candidate regulatory compound.
5. The method according to claim 1, wherein said reporter gene is selected from the group consisting of: luciferase, green fluorescent protein ("GFP"), β-galactosidase ("GAL"), chloramphenicol acetyltransferase ("CAT ").
6. 6. The method according to claim 1, wherein said reporter gene is a suppressor gene.
7. 7. The method according to claim 6, wherein said suppressor gene is l? Bsd.
8. The method according to claim 1, wherein said regulatory element of action in cis is modulated by regulatory proteins related to inflammation.
9. The method according to claim 1, wherein said cis-acting regulatory element is selected from the group consisting of AP-1, CRE,
10. 10. The method according to claim 1, wherein said immunocyte is selected from the group consisting of macrophages, CD4 + T cells and immature dendritic cells.
11. 11. A method for inhibiting the expression of a signaling pathway in an immunocyte, which consists of the following steps (a) transfecting the immunocyte with a recombinant adenovirus, wherein said recombinant adenovirus contains a suppressor gene operably linked to a regulatory element of cis action, where said cis-acting regulatory element belongs to said signaling pathway and (b) inducing the expression of said suppressor gene.
12. The method according to claim 11, wherein said signaling pathway is the signaling pathway of NFKB.
13. The method according to claim 11, wherein said suppressor gene is Bsd.
14. 14. The method according to claim 11, wherein said nanocyte is selected from the group consisting of a macrophage, a CD4 + T cell and an immature dendritic cell.