Harnessing Fc/FcRn Affinity Data from Patents with Different Machine Learning Methods.

Monoclonal antibodies are biopharmaceuticals with a very long half-life due to the binding of their Fc portion to the neonatal receptor (FcRn), a pharmacokinetic property that can be further improved through engineering of the Fc portion, as demonstrated by the approval of several new drugs. Many Fc variants with increased binding to FcRn have been found using different methods, such as structure-guided design, random mutagenesis, or a combination of both, and are described in the literature as well as in patents. Our hypothesis is that this material could be subjected to a machine learning approach in order to generate new variants with similar properties. We therefore compiled 1323 Fc variants affecting the affinity for FcRn, which were disclosed in twenty patents. These data were used to train several algorithms, with two different models, in order to predict the affinity for FcRn of new randomly generated Fc variants. To determine which algorithm was the most robust, we first assessed the correlation between measured and predicted affinity in a 10-fold cross-validation test. We then generated variants by in silico random mutagenesis and compared the prediction made by the different algorithms. As a final validation, we produced variants, not described in any patents, and compared the predicted affinity with the experimental binding affinities measured by surface plasmon resonance (SPR). The best mean absolute error (MAE) between predicted and experimental values was obtained with a support vector regressor (SVR) using six features and trained on 1251 examples. With this setting, the error on the log(KD) was less than 0.17. The obtained results show that such an approach could be used to find new variants with better half-life properties that are different from those already extensively used in therapeutic antibody development.

New recognition specificity in a plant immune receptor by molecular engineering of its integrated domain.

Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that recognize pathogen effectors. Here, we show that molecular engineering of the integrated decoy domain (ID) of an NLR can extend its recognition spectrum to a new effector. We relied for this on detailed knowledge on the recognition of the Magnaporthe oryzae effectors AVR-PikD, AVR-Pia, and AVR1-CO39 by, respectively, the rice NLRs Pikp-1 and RGA5. Both receptors detect their effectors through physical binding to their HMA (Heavy Metal-Associated) IDs. By introducing into RGA5_HMA the AVR-PikD binding residues of Pikp-1_HMA, we create a high-affinity binding surface for this effector. RGA5 variants carrying this engineered binding surface perceive the new ligand, AVR-PikD, and still recognize AVR-Pia and AVR1-CO39 in the model plant N. benthamiana. However, they do not confer extended disease resistance specificity against M. oryzae in transgenic rice plants. Altogether, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs.

Lactoferrin Retargets Human Adenoviruses to TLR4 to Induce an Abortive NLRP3-Associated Pyroptotic Response in Human Phagocytes.

Despite decades of clinical and preclinical investigations, we still poorly grasp our innate immune response to human adenoviruses (HAdVs) and their vectors. In this study, we explored the impact of lactoferrin on three HAdV types that are being used as vectors for vaccines. Lactoferrin is a secreted globular glycoprotein that influences direct and indirect innate immune response against a range of pathogens following a breach in tissue homeostasis. The mechanism by which lactoferrin complexes increases HAdV uptake and induce maturation of human phagocytes is unknown. We show that lactoferrin redirects HAdV types from species B, C, and D to Toll-like receptor 4 (TLR4) cell surface complexes. TLR4-mediated internalization of the HAdV-lactoferrin complex induced an NLRP3-associated response that consisted of cytokine release and transient disruption of plasma membrane integrity, without causing cell death. These data impact our understanding of HAdV immunogenicity and may provide ways to increase the efficacy of HAdV-based vectors/vaccines.

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences.

In order to increase the successful development of recombinant antibodies and fragments, it seems fundamental to enhance their expression and/or biophysical properties, such as the thermal, chemical, and pH stabilities. In this study, we employed a method bases on replacing the antibody framework region sequences, in order to promote more particularly single-chain Fragment variable (scFv) product quality. We provide evidence that mutations of the VH- C-C' loop might significantly improve the prokaryote production of well-folded and functional fragments with a production yield multiplied by 27 times. Additional mutations are accountable for an increase in the thermal (+19.6 °C) and chemical (+1.9 M) stabilities have also been identified. Furthermore, the hereby-produced fragments have shown to remain stable at a pH of 2.0, which avoids molecule functional and structural impairments during the purification process. Lastly, this study provides relevant information to the understanding of the relationship between the antibodies amino acid sequences and their respective biophysical properties.

Transportin-1 binds to the HIV-1 capsid via a nuclear localization signal and triggers uncoating.

The initial steps of HIV replication in host cells prime the virus for passage through the nuclear pore and drive the establishment of a productive and irreparable infection1,2. The timely release of the viral genome from the capsid-referred to as uncoating-is emerging as a critical parameter for nuclear import, but the triggers and mechanisms that orchestrate these steps are unknown. Here, we identify ß-karyopherin Transportin-1 (TRN-1) as a cellular co-factor of HIV-1 infection, which binds to incoming capsids, triggers their uncoating and promotes viral nuclear import. Depletion of TRN-1, which we characterized by mass spectrometry, significantly reduced the early steps of HIV-1 infection in target cells, including primary CD4+ T cells. TRN-1 bound directly to capsid nanotubes and induced dramatic structural damage, indicating that TRN-1 is necessary and sufficient for uncoating in vitro. Glycine 89 on the capsid protein, which is positioned within a nuclear localization signal in the cyclophilin A-binding loop, is critical for engaging the hydrophobic pocket of TRN-1 at position W730. In addition, TRN-1 promotes the efficient nuclear import of both viral DNA and capsid protein. Our study suggests that TRN-1 mediates the timely release of the HIV-1 genome from the capsid protein shell and efficient viral nuclear import.

CCSP counterbalances airway epithelial-driven neutrophilic chemotaxis.

Club cell secretory protein (CCSP) knockout mice exhibit increased airway neutrophilia, as found in chronic obstructive pulmonary disease (COPD). We therefore investigated whether treating COPD airway epithelia with recombinant human CCSP (rhCCSP) could dampen exaggerated airway neutrophilia.Control, smoker and COPD air-liquid interface (ALI) cultures exposed to cigarette smoke extract (CSE) were treated with and without rhCCSP. The chemotactic properties of the supernatants were assessed using Dunn chambers. Neutrophil chemotaxis along recombinant human interleukin 8 (rhIL8) gradients (with and without rhCCSP) was also determined. rhCCSP-rhIL8 interactions were tested through co-immunoprecipitation, Biacore surface plasmon resonance (SPR) and in silico modelling. The relationship between CCSP/IL8 concentration ratios in the supernatant of induced sputum from COPD patients versus neutrophilic airway infiltration assessed in lung biopsies was assessed.Increased neutrophilic chemotactic activity of CSE-treated ALI cultures followed IL8 concentrations and returned to normal when supplemented with rhCCSP. rhIL8-induced chemotaxis of neutrophils was reduced by rhCCSP. rhCCSP and rhIL8 co-immunoprecipitated. SPR confirmed this in vitro interaction (equilibrium dissociation constant=8 µM). In silico modelling indicated that this interaction was highly likely. CCSP/IL8 ratios in induced sputum correlated well with the level of small airway neutrophilic infiltration (r2=0.746, p<0.001).CCSP is a biologically relevant counter-balancer of neutrophil chemotactic activity. These different approaches used in this study suggest that, among the possible mechanisms involved, CCSP may directly neutralise IL8.

Binding analysis between monomeric ß-casein and hydrophobic bioactive compounds investigated by surface plasmon resonance and fluorescence spectroscopy.

ß-Casein, a phosphoprotein representing 37% of the bovine milk caseins, has specific features promoting its application as a nanocarrier for hydrophobic bioactives. In this study, the interactions of ß-casein with curcumin and vitamin D3 under the same physico-chemical conditions were investigated. The interaction kinetics have been studied by surface plasmon resonance (SPR) and fluorescence spectroscopy. The KD value for curcumin-ß-casein interaction has been successfully evaluated (4.1 ±â€¯0.7 × 10-4 M) using SPR by fitting data to a 1:1 Langmuir interaction model. Conversely, the SPR responses obtained for vitamin D3 show that the interactions between this hydrophobic compound and the ß-casein immobilized on the sensor chip were below the sensitivity of the SPR apparatus. Moreover, the fluorescence quenching data show that curcumin has higher affinity to ß-casein (KA = 23.5 ±â€¯1.9 × 104 M-1) than vitamin D3 (KA = 5.8 ±â€¯1.1 × 104 M-1).

PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ.

PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus.

Thrombospondin-1 (TSP-1), a new bone morphogenetic protein-2 and -4 (BMP-2/4) antagonist identified in pituitary cells.

Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.

Nanofluidic Fluorescence Microscopy (NFM) for real-time monitoring of protein binding kinetics and affinity studies.

Kinetic monitoring of protein interactions offers insights to their corresponding functions in cellular processes. Surface plasmon resonance (SPR) is the current standard tool used for label-free kinetic assays; however, costly and sophisticated setups are required, decreasing its accessibility to research laboratories. We present a cost-effective nanofluidic-based immunosensor for low-noise real-time kinetic measurement of fluorescent-labeled protein binding. With the combination of fluorescence microscopy and reversed buffer flow operation, association and dissociation kinetics can be accessed in one single experiment without extra buffer loading step, which results in a simplified operation and reduced time of analysis compared to typical microfluidic immunoassays. Kinetic constants of two representative protein-ligand binding pairs (streptavidin/biotin; IgG/anti-IgG) were quantified. The good agreement of extracted rate constants with literature values and analogous SPR measurements indicates that this approach is applicable to study protein interactions of medium- and high-affinities with a limit of detection down to 1 pM, regardless of the analyte size.

Increment in Drug Loading on an Antibody-Drug Conjugate Increases Its Binding to the Human Neonatal Fc Receptor in Vitro.

Antibody-drug conjugates, such as brentuximab vedotin (BTXv), are an innovative category of monoclonal antibodies. BTXv is bioconjugated via the chemical reduction of cysteine residues involved in disulfide bonds. Species of BTXv containing zero, two, four, six, or eight vedotin molecules per antibody coexist in the stock solution. We investigated the influence of drug loading on the binding of the antibody to FcRn, a major determinant of antibody pharmacokinetics in humans. We developed a hydrophobic interaction chromatography (HIC) method for separating the different species present in the stock solution of BTXv, and we purified and characterized the collected species before use. We assessed the binding of these different species to FcRn in a cellular assay based on flow cytometry and surface plasmon resonance. HIC separated the different species of BTXv and allowed their collection at adequate levels of purity. Physicochemical characterization showed that species with higher levels of drug loading tended to form more aggregates. FcRn binding assays showed that the most conjugated species, particularly those with saturated loading, interacted more strongly than unconjugated BTXv with the FcRn.

A method to confer Protein L binding ability to any antibody fragment.

Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) domain. Not all kappa chains are recognized, however, and the lack of CL can reduce the interaction. From a scFv composed of IGKV10-94 according to IMGT®, it is possible, with several mutations, to transfer the motif from the IGKV12-46 naturally recognized by the PpL, and, with the single mutation T8P, to confer PpL recognition with a higher affinity. A second mutation S24R greatly improves the affinity, in particular by modifying the dissociation rate (kd). The equilibrium dissociation constant (KD) was measured at 7.2 10(-11) M by surface plasmon resonance. It was possible to confer PpL recognition to all kappa chains. This protein interaction can be modulated according to the characteristics of scFv (e.g., stability) and their use with conjugated PpL. This work could be extrapolated to recombinant monoclonal antibodies, and offers an alternative for protein A purification and detection.

Fragment-based identification of a locus in the Sec7 domain of Arno for the design of protein-protein interaction inhibitors.

By virtual screening using a fragment-based drug design (FBDD) approach, 33 fragments were selected within small pockets around interaction hot spots on the Sec7 surface of the nucleotide exchange factor Arno, and then their ability to interfere with the Arno-catalyzed nucleotide exchange on the G-protein Arf1 was evaluated. By use of SPR, NMR, and fluorescence assays, the direct binding of three of the identified fragments to Arno Sec7 domain was demonstrated and the promiscuous aggregate behavior evaluated. Then the binding mode of one fragment and of a more active analogue was solved by X-ray crystallography. This highlighted the role of stable and transient pockets at the Sec7 domain surface in the discovery and binding of interfering compounds. These results provide structural information on how small organic compounds can interfere with the Arf1-Arno Sec7 domain interaction and may guide the rational drug design of competitive inhibitors of Arno enzymatic activity.

Abrogation of de novo lipogenesis by stearoyl-CoA desaturase 1 inhibition interferes with oncogenic signaling and blocks prostate cancer progression in mice.

Increased de novo fatty acid (FA) synthesis is one hallmark of tumor cells, including prostate cancer. We present here our most recent results showing that lipid composition in human prostate cancer is characterized by an increased ratio of monounsaturated FA to saturated FA, compared with normal prostate, and evidence the overexpression of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1) in human prostate cancer. As a new therapeutic strategy, we show that pharmacologic inhibition of SCD1 activity impairs lipid synthesis and results in decreased proliferation of both androgen-sensitive and androgen-resistant prostate cancer cells, abrogates the growth of prostate tumor xenografts in nude mice, and confers therapeutic benefit on animal survival. We show that these changes in lipid synthesis are translated into the inhibition of the AKT pathway and that the decrease in concentration of phosphatidylinositol-3,4,5-trisphosphate might at least partially mediate this effect. Inhibition of SCD1 also promotes the activation of AMP-activated kinase and glycogen synthase kinase 3alpha/beta, the latter on being consistent with a decrease in beta-catenin activity and mRNA levels of various beta-catenin growth-promoting transcriptional targets. Furthermore, we show that SCD1 activity is required for cell transformation by Ras oncogene. Together, our data support for the first time the concept of targeting the lipogenic enzyme SCD1 as a new promising therapeutic approach to block oncogenesis and prostate cancer progression.

miR-143 interferes with ERK5 signaling, and abrogates prostate cancer progression in mice.

BACKGROUND: Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. RESULTS: Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5) activity. We show here that ERK5 is a miR-143 target in prostate cancer. CONCLUSIONS: miR-143 is as a new target for prostate cancer treatment.

Anti-inflammatory properties of desipramine and fluoxetine.

BACKGROUND: Antidepressants are heavily prescribed drugs and have been shown to affect inflammatory signals. We examined whether these have anti-inflammatory properties in animal models of septic shock and allergic asthma. We also analysed whether antidepressants act directly on peripheral cell types that participate in the inflammatory response in these diseases. METHODS: The antidepressants desipramine and fluoxetine were compared in vivo to the glucocorticoid prednisolone, an anti-inflammatory drug of reference. In a murine model of lipopolysaccharides (LPS)-induced septic shock, animals received the drugs either before or after injection of LPS. Circulating levels of tumour necrosis factor (TNF)-alpha and mortality rate were measured. In ovalbumin-sensitized rats, the effect of drug treatment on lung inflammation was assessed by counting leukocytes in bronchoalveolar lavages. Bronchial hyperreactivity was measured using barometric plethysmography. In vitro production of TNF-alpha and Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) from activated monocytes and lung epithelial cells, respectively, was analysed by immunoassays. Reporter gene assays were used to measure the effect of antidepressants on the activity of nuclear factor-kappaB and activator protein-1 which are involved in the control of TNF-alpha and RANTES expression. RESULTS: In the septic shock model, all three drugs given preventively markedly decreased circulating levels of TNF-alpha and mortality (50% mortality in fluoxetine treated group, 30% in desipramine and prednisolone treated groups versus 90% in controls). In the curative trial, antidepressants had no statistically significant effect, while prednisolone still decreased mortality (60% mortality versus 95% in controls). In ovalbumin-sensitized rats, the three drugs decreased lung inflammation, albeit to different degrees. Prednisolone and fluoxetine reduced the number of macrophages, lymphocytes, neutrophils and eosinophils, while desipramine diminished only the number of macrophages and lymphocytes. However, antidepressants as opposed to prednisolone did not attenuate bronchial hyperreactivity. In vitro, desipramine and fluoxetine dose-dependently inhibited the release of TNF-alpha from LPS-treated monocytes. In lung epithelial cells, these compounds decreased TNF-alpha-induced RANTES expression as well as the activity of nuclear factor-kappaB and activator protein-1. CONCLUSION: Desipramine and fluoxetine reduce the inflammatory reaction in two animal models of human diseases. These antidepressants act directly on relevant peripheral cell types to decrease expression of inflammatory mediators probably by affecting their gene transcription. Clinical implications of these observations are discussed.

Differential regulation of RANTES and IL-8 expression in lung adenocarcinoma cells.

In lung adenocarcinoma, expression of Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) is a predictor of survival while that of interleukin (IL)-8 is associated with a poor prognosis. In several models, tumorigenesis is abolished by RANTES, while it is facilitated by IL-8. We studied the regulation of RANTES and IL-8 expression in A549 lung adenocarcinoma cells. The effects of tumor necrosis factor (TNF)-alpha and regulators of protein kinases C (PKC)alpha/beta were tested because these have been shown to modulate cancer development and progression. TNF-alpha stimulated expression of both chemokines, while the PKCalpha/beta activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced only expression of IL-8 and inhibited TNF-alpha-induced RANTES expression. The PKCalpha/beta inhibitor Gö 6976 increased TNF-alpha-induced RANTES production and prevented its down-regulation by TPA. In contrast, it decreased TNF-alpha or TPA-induced IL-8 release. The differential regulation of RANTES and IL-8 expression was further analyzed. Site-directed mutagenesis indicated that regulation of RANTES promoter activity required two nuclear factor (NF)-kappaB response elements but not its activator protein (AP)-1 binding sites. An AP-1 and a NF-kappaB recognition sites were necessary for full induction of IL-8 promoter activity by TNF-alpha and TPA. Moreover, electrophoretic mobility shift assays demonstrated that NF-kappaB response elements from the RANTES promoter were of lower affinity than that from the IL-8 promoter. Immunoblotting experiments showed that TPA was more potent than TNF-alpha to induce in a PKCalpha/beta dependent manner the p44/p42 mitogen-activated protein kinases (MAPK) signaling cascade which controls AP-1 activity. Conversely, TPA inhibited TNF-alpha-induced NF-kappaB signaling and was a weak activator of this pathway. Thus, TPA did not sufficiently activate NF-kappaB to increase transcription through the low affinity NF-kappaB binding sites on RANTES promoter and its inhibitory effect on TNF-alpha-induced NF-kappaB signaling resulted in a reduced transcription rate. On IL-8 promoter, increased transcription through the high affinity NF-kappaB binding site occurred even with poorly activated NF-kappaB and the functional AP-1 response element compensated any loss of transcription rate. These data provide a mechanistic insight into the differential regulation of IL-8 and RANTES expression by PKCalpha/beta in lung adenocarcinoma cells.

Overexpression of the human glucocorticoid receptor alpha and beta isoforms inhibits AP-1 and NF-kappaB activities hormone independently.

The human glucocorticoid receptor isoforms GRalpha and GRbeta are generated by alternative splicing. Upon hormone binding, GRalpha regulates positively or negatively transcription. In particular, it represses numerous genes encoding pro-inflammatory mediators by inhibiting the transcription factors activator protein (AP)-1 and nuclear factor (NF)-kappaB. The observation that GRbeta, which does not bind the hormone, may act as a dominant negative receptor is subject to controversy. Because GRbeta must be more abundant than GRalpha to act as such, we evaluated the relative amounts of GRalpha and GRbeta in COS-1, A549 and HeLa cells using a monoclonal antibody that recognises the two isoforms equally well on western blots. Messenger RNA levels of GRalpha and GRbeta were compared by reverse transcriptase polymerase chain reaction analysis. To gain insight into the possible function of GRbeta, we examined the ability of overexpressed GRbeta to alter transcription of glucocorticoid, AP-1 and NF-kappaB inducible reporter genes using transient transfection in COS-1 and A549 cells. Subcellular localisation of GRbeta was determined in A549 cells by immunofluoresence microscopy. Data indicate that GRalpha is the predominant endogenous isoform in A549 and HeLa cells. GRbeta became the major form after transfection with the corresponding expression vector and translocated into cell nuclei even in the absence of hormone. Overexpression of GRbeta inhibited glucocorticoid-induced transcription markedly in COS-1 cells but weakly in A549 cells. We found that GRbeta did not act as a dominant negative modulator of GRalpha for repression of AP-1 and NF-kappaB activities. In fact, both GRbeta and GRalpha inhibited hormone-independently these activities by 25-60%. This property was not shared by the closely related mineralocorticoid receptor. Our results suggest that overexpression of either GRalpha or GRbeta may have an anti-inflammatory effect.