Patent Application: US-22933305-A

Abstract:
described are methods of treating allergic disorders and compositions for use therein . the methods comprise administering an allergen and one or more medicaments . these medicaments are compounds that inhibit the transcription of genes involved in the initiation of innate and specific immunity , thereby promoting the development of tolerance to these allergens , through inhibition of the nf - κb and / or the mapk / ap - 1 signal transduction pathway . in another embodiment , the use of dna vaccines is disclosed that incorporates a gene encoding one or more allergen sequences or fragments thereof , in combination with genes encoding proteins that inhibit the activation of the nf - κb and / or the mapk / ap - 1 pathway or in combination with small interfering rna sequences or anti - sense sequences that inhibit the expression of np - κb and / or ap - 1 proteins .

Description:
the nf - κb family of transcription factors has a central role in coordinating the expression of a wide variety of genes that control immune and inflammatory responses , including cytokines , chemokines , cell - adhesion molecules , co - stimulatory molecules , complement factors and anti - apoptotic factors ( mckay et al ., 1999 ). mammalian nf - κb family members include rela ( p65 ), nf - κb1 ( p50 ; p150 ), nf - κb2 ( p52 ; p100 ), crel and relb . importantly , experiments with gene - deleted mice have proved that nf - κb1 p50 , rela and crel are essential in the innate immune function of dc . nf - κb proteins are present in the cytoplasm in association with inhibitory proteins that are known as inhibitors of nf - κb ( iκbs ). the iκb family of proteins consists of iκbα , iκbβ , iκbε and bcl - 3 ( li , nrdd ). an essential step in the activation of innate immune cells by pathogens , stress molecules and pro - inflammatory cytokines is the degradation of iκb and release of nf - κb and its subsequent phosphorylation allowing nf - κb proteins to translocate to the nucleus and bind to their cognate dna binding sites to regulate the transcription of large numbers of genes . a crucial regulatory step in the degradation of iκbs is the signal - induced phosphorylation of iκb at specific amino - terminal serine residues , which is mediated by serine - specific iκb kinases ( ikk ). the serine phosphorylated iκb is then ubiquitinilated and degraded by the proteasome . the ikk complex consists of several proteins , the main ones being ikk1 ( ikkγ ), ikk2 and the regulatory subunit nf - κb essential modulator ( nemo , also known as ikkγ ). inhibition of nf - κb activation can be accomplished by several strategies including , but not limited to , direct targeting the dna - binding activity of individual nf - κb proteins using small molecules or decoy oligonucleotides ; treatment with cell membrane - permeable non - degradable iκbα , - β or - ε mutant protein ( s ); blocking the nuclear translocation of nf - κb dimers by inhibiting the nuclear import system ; stabilizing iκbα , - β or - ε protein ( s ) by developing ubiquitylation and proteasome inhibitors ; targeting signaling kinases such as ikk using small - molecule inhibitors ( li et al ., 2002 ); and treatment with cell membrane - permeable dominant negative ikk protein . several drugs that are used to treat inflammatory diseases have effects on nf - κb activity such as glucocorticosteroids ( gcs ), aspirin and other anti - inflammatory drugs . although these drugs do not target nf - κb specifically , parts of their pharmacologic effects are due to inhibition of nf - κb activity . besides these drugs , many compounds have been described in literature as inhibitors of nf - κb activation , such as , for example , anti - oxidants , proteasome and protease inhibitors , iκb phosphorylation and / or degradation inhibitors and miscellaneous inhibitors ( table 1 ). it will be clear to a person skilled in the art that functional analogues to the compounds as listed in table 1 can also be used to inhibit nf - κb activation . a functional analogue exhibits the same inhibitory activity of nf - κb activation in kind if not in amount . mammals express at least four distinctly regulated groups of mitogen - activated protein kinases ( mapks ), erk - 1 / 2 , erk5 , jnk1 / 2 / 3 and p38α / β / γ / δ , that have been shown to regulate several physiological and pathological cellular phenomena , including inflammation , apoptotic cell death , oncogenic transformation , tumor cell invasion and metastasis ( herlaar et al ., 1999 ). upon cellular stimulation , a kinase cascade is initiated that ultimately leads to altered gene expression and consequently a biological response . the three main kinases in the mapk cascades are the mapk kinase kinase ( mkkk ), mapk kinase ( mkk ) and mapk . in total , 12 mapk isoforms have been identified that can phosphorylate and activate a large range of substrates , including transcription factors and kinases . p38mapk and jnk are stress - activated protein kinases that mediate responses to cellular stress factors such as uv light and oxidative stress . a wide variety of inflammatory mediators , such as cytokines , activate p38 mapk in immune - and inflammatory cells . to date , several specific mapk inhibitors have been developed in particular targeting p38 mapk . the pyridinylimidazole compounds , exemplified by sb 203580 , have been demonstrated to be selective inhibitors of p38 mapk . this compound specifically inhibits p38α , β and β2 mapk and has shown activity in a variety of animal models of acute and chronic inflammation . other small molecule compounds that inhibit p38 mapk are vx - 745 ( vertex pharmaceuticals ), rwj67657 ( johnson & amp ; johnson ) and hep 689 ( leo pharmaceuticals ). interestingly , sb 203580 has been shown to inhibit the maturation of dendritic cells . other compounds that have been shown to inhibit dendritic cell maturation through inhibition of p38 mapk are the anti - inflammatory sesquiterpene lactone parthenolide ( ptl ) and the cytokine il - 10 . in contrast , inhibition of the erk mapk pathway by the selective inhibitors pd98059 and u0126 , has been shown to enhance phenotypic and functional maturation of dendritic cells . little is known about the role of jnk mapk in the regulation of innate - and adaptive immune responses . the jnk inhibitor sp600125 has been shown to inhibit the induction of il - 18 production by macrophages and the signaling of the t1 / st2 , a cell membrane receptor that is selectively expressed on th2 lymphocytes . mapks are upstream regulators of ap - 1 . the transcription factor family activator protein 1 ( ap - 1 ) is formed by heterodimeric complexes of a fos protein ( c - fos , fra - 1 , fra - 2 , fosb and fosb2 ) with a jun protein ( c - jun , junb and jund ) or a homodimer between two jun proteins ( foletta et al ., 1998 ). ap - 1 regulates many of the genes up - regulated during immune - and inflammatory responses . the most well - known repressor of the transcription factor ap - 1 are glucocorticoids . together with the inhibition nf - κb activation by glucocorticoids , these are the major mechanisms for the anti - inflammatory effects of this drug class ( mckay et al ., 1999 ). activation of gene transcription by ap - 1 can also be inhibited by decoy oligonucleotides . it will be clear to a person skilled in the art that functional analogues to the compounds as mentioned above and used for the inhibition of the mapk / ap - 1 pathway can also be used to inhibit mapk / ap - 1 pathway activation . a functional analogue exhibits the same inhibitory activity of the mapkiap - 1 pathway in kind , if not in amount . the activation of the nf - κb and / or mapk / ap - 1 pathways can also be prevented by interference with “ co - stimulation ” molecules or locally produced activating mediators by ( i ) blocking of nf - κb and / or mapk / ap - 1 - activating mediators , including , but not limited to , cytokines such as il - 1 , - 2 , - 12 , - 15 , - 17 , - 18 , lif , and members of the tnf super - family such as fas ligand , gitr ligand , thank , rank ligand ( also called trance or opgl ), tnfα and tnfβ or blocking their specific cell membrane receptors , or ( ii ) blocking prr including , but not limited to , toll - like receptors , lectin receptors or nods , or ( iii ) prevention of oxidative stress using anti - oxidants , or ( iv ) blocking extra - cellular heat - shock proteins or their cell membrane receptors , or ( v ) blocking purinergic receptors , in particular , those expressed on apcs . nf - κb activation , in particular in apcs , can also be inhibited by compounds that increase intracellular levels of cyclic amp including , but not limited to , β2 - adrenoceptor agonists , prostanoid ep2 - or dp receptor agonists or phosphodiesterase iv inhibitors . inhibition of nf - κb and mapk / ap − 1 signal transduction pathways by ppar activation recently , an interesting family of nuclear hormone receptors have emerged called peroxisome proliferator - activated receptors ( ppars ) that , upon ligation , exert potent inhibitory effects on the transcription factors nf - κb and ap - 1 ( daynes et al ., 2002 ; hihi et al ., 2002 ). so far , three ppar isoforms have been identified pparα , pparβ / δ and pparγ with a high degree of sequence and structural homology . ppars share the property of forming heterodimers with another nuclear receptor of the same subgroup , the 9 - cis - retinoic acid receptor ( rxr ), which appears to be essential for their biological function . various types of fatty acids and eicosanoids can bind to and activate ppars , with some degree of isoform specificity ( daynes et al ., 2002 ; hihi et al ., 2002 ). pparα can be activated by α - linoleic -, γ - linoleic -, arachidonic - and eicosapentaenoic acids and by medium - chain saturated and monounsaturated fatty acids such as palmitic and oleic acids . pparα can be activated selectively by ltb4 and 8 ( s ) hete . pparγ is activated by α - linoleic -, γ - linoleic -, arachidonic - and eicosapentaenoic acids , although these endogenous ligands are weak activators . pparγ is best stimulated by 9 - hode , 13 - hode and 15dpgj2 and by the synthetic compound rosiglitazone and thiazolidinedione class of drugs . pparβ / δ can be activated by some saturated , monounsaturated and unsaturated fatty acids , and by various eicosanoids including pga1 and pgd2 and prostacyclin or a stable synthetic form . interestingly , pparα and pparγ are expressed in antigen - presenting cells ( monocytes , macrophages , dendritic cells and b - cells ) and can play an important role in down - regulation of nf - κb and ap - 1 activity ( daynes et al ., 2002 ; nencioni et al ., 2002 ). the standard dna vaccine consists of the specific gene ( s ) of interest cloned into a bacterial plasmid engineered for optimal expression in eukaryotic cells . essential features include a strong promoter for optimal expression in mammalian cells , an origin of replication allowing growth in bacteria , a bacterial antibiotic - resistance gene and incorporation of polyadenylation sequences to stabilize mrna transcripts . moreover , dna vaccines also contain specific nucleotide sequences that play a critical role in the immunogenicity of these vaccines . in case of allergen vaccination using dna vaccines , the plasmid contains nucleotide sequences encoding one or more allergens or allergen fragments containing at least one t - cell epitope sequence . allergens for use in the invention include , but are not limited to , the list available on the world - wide web at http :// www . allergen . org / list . htm . it has been shown that immune responses induced by dna vaccination are mediated by apcs , in particular , dcs migrating from the site of vaccination to the draining lymph nodes . the dcs are either directly transfected or take up secreted protein from other transfected cells , i . e ., myocytes . fusion of the allergen or allergen fragment to an igg fc fragment improves the secretion of the encoding allergen and the subsequent targeting to and uptake by apcs . targeting of dna vaccines to apcs , in particular dcs , may be obtained by using particular viral vectors including , but not limited to , herpes virus , vaccinia virus , adenovirus , influenza virus , retroviruses and lentiviruses ( jenne et al ., 2001 ). second generation dna vaccines are also being developed that introduce not only a gene encoding the target antigen , but also a gene encoding some other factor capable of inducing an altered immune response . within the present invention , the plasmid comprising a t - cell epitope can be combined with genes encoding proteins that inhibit the activation of the nf - κb pathway , including , but not limited to , ( non - degradable ) iκb proteins or dominant negative forms of ikk proteins or nf - κb proteins and / or genes encoding proteins that inhibit the activation of the mapk / ap - 1 pathway , including , but not limited to , dominant negative forms of critical proteins leading to the activation of fos and / or jun proteins such as p38 mapk or dominant negative forms of fos and / or jun proteins . in another embodiment , the plasmid comprising a t - cell epitope can be combined with small interfering rna sequences or anti - sense sequences that inhibit the expression of ikk , nf - κb , p38 mapk or ap - 1 proteins . of course , the effects of inhibiting or preventing the production of a co - stimulator molecule in an antigen - presenting cell , when the antigen - presenting cell is contacted with an allergen , can also be studied and assessed on isolated cells in vitro . the effects of a compound on the production of a co - stimulator molecule can thus be tested in vitro and a selection can be made as to what compound is most suitable for inhibiting the production of a co - stimulator molecule by an antigen - presenting cells . therefore , the present invention also teaches a method to inhibit and / or prevent the production of a co - stimulator molecule in an antigen - presenting cell in the presence of an allergen , wherein the production of a co - stimulator molecule is inhibited and / or prevented by inhibiting the nf - κb and / or the mapk / ap - 1 signal - transducing pathways in the antigen - presenting cell . in a recent who position paper , allergen vaccination or immunotherapy is defined as the practice of administering gradually increasing quantities of an allergen extract to an allergic subject to ameliorate the symptoms associated with subsequent exposure to the causative allergen ( bousquet et al ., 1998 ). in the present invention , we describe novel forms of allergen vaccination that offer significant advantages over current allergen immunotherapy practice . allergens for use in the invention include , but are not limited to , the list available on the world - wide web at http :// www . allergen . org / list . htm . the allergen used can be an allergen extract such as house - dust mite or pollen or fragments thereof containing at least one t - cell epitope or an entire or partial recombinant allergen protein such as der p1 containing at least one cell epitope . the preferred route of administration is subcutaneous injection , however , other routes , such as nasal , oral or sublingual application , can be effective as well . another embodiment is the use of dna vaccines that incorporate a gene encoding the entire or partial allergen sequence and containing at least one t - cell epitope sequence ( walker et al ., 2001 ). an allergen vaccination course usually involves a build - up phase ( increasing allergen dose ) and a maintenance phase ( maximum dosage of the allergen ) in which the allergen is administered with a 1 to 2 month interval . the duration of allergen vaccination required to maintain improvement in clinical symptoms has been advised to 3 to 5 years of therapy ( bousquet et at ., 1998 ). allergen vaccination is rarely started before the age of 5 years . when started early in the disease process , allergen vaccination may modify the progression of the disease . novel allergen vaccination strategies consist of the treatment with one or more compounds that inhibit the nf - κb pathway and / or the mapk / ap - 1 pathway at the time of allergen injection . this / these compound ( s ) may be co - injected subcutaneously together with the allergen or given separately by systemic , enteral , or parenteral administration . a non - exhaustive list of inhibitors of nf - κb activation is provided in table 1 . in case of dna vaccination , the plasmid comprising a t - cell epitope can be combined with genes that inhibit the nf - κb and / or the mapk / ap - 1 pathway . the methods provided herein are suitable for treating any allergic disorders including , but not limited to , rhinitis , food allergy , urticaria , atopic dermatitis and asthma . animals . animal care and use were performed in accordance with the guidelines of the dutch committee of animal experiments . specific pathogen - free male balb / c mice ( 5 to 6 weeks old ) were purchased from charles river ( maastricht , the netherlands ) and housed in macrolon cages in a laminar flow cabinet and provided with food and water ad libitum . sensitization , treatment and challenge . mice ( 6 to 8 weeks old ) were sensitized intraperitoneally ( i . p .) on days 0 and 7 with 10 μg ovalbumin ( ova , grade v , sigma - aldrich ) in 0 . 1 ml alum ( pierce , rockford , ill .). two weeks after the last sensitization , the mice were divided into six groups . the sham - immunotherapy and the ova - immunotherapy groups were treated with three s . c . injections of , respectively , 0 . 2 ml pyrogen - free saline ( b . braun , melsungen , germany ) or 1 mg ova in 0 . 2 ml pyrogen - free saline on alternate days . in three groups , ova - immunotherapy was co - injected with 0 . 1 μg , 0 . 03 μg or 0 . 01 μg 1α , 25 - dihydroxyvitamin d3 ( 1α , 25 ( oh ) 2 vitd3 ), a selective inhibitor of nf - κb . one group was treated with sham - immunotherapy and combined with co - injection of 0 . 1 μg 1α , 25 ( oh ) 2 vitd3 . ten days after treatment , mice were exposed to three ova inhalation challenges ( 10 mg / ml in saline ) for 20 minutes every third day . in a second series of experiments , ova - immunotherapy , as described above , was carried out using a sub - optimal amount of 100 μg ova in saline for ova - immunotherapy . ova - immunotherapy was either given alone or in combination with 0 . 01 μg 1α , 25 ( oh ) 2 vitd3 . sham - immunotherapy alone or in combination with 0 . 01 μg 1α , 25 ( oh ) 2 vitd3 served as control groups . measurement of airway responsiveness in vivo . airway responsiveness to methacholine was measured after treatment but before ova challenge ( pre - measurement ) and 24 hours after the last ova challenge . airway responsiveness was measured in conscious , unrestrained mice using barometric whole - body plethysmography by recording respiratory pressure curves ( buxco , emka technologies , paris , france ) in response to inhaled methacholine ( acetyl - β - methylcholine chloride , sigma - aldrich ). airway responsiveness was expressed in enhanced pause ( penh ), as described in detail previously ( deurloo et al ., 2001 ). briefly , mice were placed in a whole - body chamber and basal readings were determined for three minutes . aerosolized saline , followed by doubling concentrations of methacholine ( ranging from 3 . 13 - 25 mg / ml in saline ), were nebulized for three minutes , and readings were determined for three minutes after each nebulization . determination of ova - specific ige levels in serum . from each mouse , serum was obtained after treatment but before ova challenge ( pre - measurement ) by a small incision in the tail vein . after measurement of airway responsiveness in vivo , mice were sacrificed by i . p . injection of 1 ml 10 % urethane in saline and were bled by cardiac puncture . subsequently , serum was collected and stored at − 70 ° c . until analysis . serum levels of ova - specific ige were measured by sandwich elisa as described previously ( deurloo et al ., 2001 ). analysis of the cellular composition of the bronchoalveolar lavage fluid . bronchoalveolar lavage ( bal ) was performed immediately after bleeding of the mice by lavage of the airways through a tracheal cannule five times with 1 ml saline ( 37 ° c .). cells in the balf were centrifuged and resuspended in cold pbs . the total number of cells in the bal was determined using a bürker - türk counting chamber ( karl hecht assistant kg , sondheim / röhm , germany ). for differential bal cell counts , cytospin preparations were made ( 15 × g , five minutes , 4 ° c ., kendro heraues instruments , asheville , n . c .). next , cells were fixed and stained with diff - quick ( dade a . g ., düdingen , switzerland ). per cytospin , 200 cells were counted and differentiated into mononuclear cells , eosinophils , and neutrophils by standard morphology and staining characteristics . statistical analysis . all data are expressed as mean ± standard error of mean ( sem ). the airway dose - response curves to methacholine were statistically analyzed by a general linear model of repeated measurements followed by post - hoc comparison between groups . data were log transformed before analysis to equalize variances in all groups . all other data were analyzed using a student &# 39 ; s t test ( 2 - tailed , homosedastic ). results were considered statistically significant at the p & lt ; 0 . 05 level . airway responsiveness in vivo . no significant differences between all six groups were observed in airway responsiveness to methacholine after treatment but prior to ova challenge ( fig1 a ). ova - sensitized balb / c mice that received sham - immunotherapy displayed significant airway hyper - responsiveness ( ahr ) to methacholine after ova inhalation challenge as compared to before challenge . mice that received ova - immunotherapy displayed significant ahr to methacholine after ova challenge as compared to before challenge . however , ova - immunotherapy partially reduced ( p & lt ; 0 . 05 ) ahr to methacholine as compared to sham - treated mice . interestingly , co - injection of 1α , 25 ( oh ) 2 vitd3 , at all doses used , with ova - immunotherapy significantly potentiated the reduction of ahr to methacholine compared to ova - it alone . co - injection of 1α , 25 ( oh ) 2 vitd3 with sham - immunotherapy did not affect ahr . it can be concluded that co - injection of the selective nf - κb inhibitor 1α , 25 ( oh ) 2 vitd3 potentiates the suppressive effect of ova - immunotherapy on ahr . ova - specific ige levels in serum . ova - sensitized balb / c mice that received sham - immunotherapy showed a significant increase in serum levels of ova - specific ige after ova inhalation challenge as compared to before challenge ( fig2 ). in mice that received ova - immunotherapy , serum ova - specific ige levels were significantly reduced after ova challenge as compared to sham - treated ova - challenged mice . co - injection of 0 . 01 μg 1α , 25 ( oh ) 2 vitd3 with ova - immunotherapy significantly increased the reduction of serum ige levels as compared to ova - immunotherapy alone . co - injection of 1α , 25 ( oh ) 2 vitd3 with sham - immunotherapy did not affect serum ige levels as compared to sham - immunotherapy alone . it can be concluded that co - injection of the selective nf - κb inhibitor 1α , 25 ( oh ) 2 vitd3 , at 0 . 01 μg , potentiates the suppression of serum ova - specific ige levels after ova - immunotherapy . cellular composition of the bronchoalveolar lavage fluid . in balb / c mice , no eosinophils are present in balf prior to ova inhalation . ova - sensitized balb / c mice that received sham - immunotherapy showed balf eosinophilia after ova inhalation challenge ( fig3 ). after ova challenge of mice that received ova - immunotherapy , the number of balf eosinophils were significantly reduced as compared to sham - treated mice . co - injection of 0 . 01 μg 1α , 25 ( oh ) 2 vitd3 with ova - immunotherapy significantly increased the reduction of balf eosinophil numbers as compared to ova - immunotherapy alone . co - injection of 1α , 25 ( oh ) 2 vitd3 with sham - immunotherapy did not significantly affect bal eosinophil number . it can be concluded that co - injection of the selective nf - κb inhibitor 1α , 25 ( oh ) 2 vitd3 , at 0 . 01 μg , potentiates the suppression of balf eosinophilia after ova - immunotherapy . collectively , it is concluded that the selective nf - κb inhibitor 1α , 25 ( oh ) 2 vitd3 is able to potentiate the beneficial effect of ova - immunotherapy on airway hyper - responsiveness , serum ige levels and airway eosinophilia , all important hallmarks of human asthma . in the results described herein , the effect of co - injection of the selective nf - κb inhibitor 1α , 25 ( oh ) 2 vitd3 with ova - immunotherapy was most pronounced with regards to potentiation of the suppression of ahr . the potentiation of the suppression of balf eosinophilia was less pronounced because the amount of ova used for ova - immunotherapy already induced a strong , almost maximal , suppression of balf eosinophil numbers ( fig3 ). therefore , we examined the effect of co - injection of 0 . 01 μg 1α , 25 ( oh ) 2 vitd3 with a sub - optimal amount of ova ( 100 μg ) for ova - immunotherapy . cellular composition of the bronchoalveolar lavage fluid . in balb / c mice , no eosinophils are present in balf prior to ova inhalation . ova - sensitized balb / c mice that received sham - immunotherapy showed balf eosinophilia after ova inhalation challenge ( fig4 ). after ova challenge of mice that received ova - immunotherapy ( 100 μg ), the number of balf eosinophils were significantly reduced as compared to sham - treated mice . co - injection of 0 . 01 μg 1α , 25 ( oh ) 2 vitd3 with a sub - optimal dose of ova - 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