Comparative angiotensin II receptor blockade in healthy volunteers: the importance of dosing.

OBJECTIVES: We have reported previously that 80 mg valsartan and 50 mg losartan provide less receptor blockade than 150 mg irbesartan in normotensive subjects. In this study we investigated the importance of drug dosing in mediating these differences by comparing the AT(1)-receptor blockade induced by 3 doses of valsartan with that obtained with 3 other antagonists at given doses. METHODS: Valsartan (80, 160, and 320 mg), 50 mg losartan, 150 mg irbesartan, and 8 mg candesartan were administered to 24 healthy subjects in a randomized, open-label, 3-period crossover study. All doses were given once daily for 8 days. The angiotensin II receptor blockade was assessed with two techniques, the reactive rise in plasma renin activity and an in vitro radioreceptor binding assay that quantified the displacement of angiotensin II by the blocking agents. Measurements were obtained before and 4 and 24 hours after drug intake on days 1 and 8. RESULTS: At 4 and 24 hours, valsartan induced a dose-dependent "blockade" of AT(1) receptors. Compared with other antagonists, 80 mg valsartan and 50 mg losartan had a comparable profile. The 160-mg and 320-mg doses of valsartan blocked AT(1) receptors at 4 hours by 80%, which was similar to the effect of 150 mg irbesartan. At trough, however, the valsartan-induced blockade was slightly less than that obtained with irbesartan. With use of plasma renin activity as a marker of receptor blockade, on day 8, 160 mg valsartan was equivalent to 150 mg irbesartan and 8 mg candesartan. CONCLUSIONS: These results show that the differences in angiotensin II receptor blockade observed with the various AT(1) antagonists are explained mainly by differences in dosing. When 160-mg or 320-mg doses were investigated, the effects of valsartan hardly differed from those obtained with recommended doses of irbesartan and candesartan.

Transcriptional Activation and Receptor Dimerization is Affected by Mutations in the NR2E3 Ligand-Binding Domain

Purpose: Mutations in the ligand-binding domain (LBD) of NR2E3 cause recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS), Goldmann-Favre syndrome (GFS) and clumped pigmentary retinal degeneration (CPRD). In addition to ligand binding, the LBD contains also essential amino acid sequences for the oligomerization of nuclear receptors. The aim of our studies is to characterize the impact of mutations in the LBD on receptor oligomerization and transcriptional activity of NR2E3. Methods: The different NR2E3 mutants were generated by QuickChange mutagenesis and analyzed in 293T-based transactivation studies and BRET2 (bioluminescence resonance electron transfer) assays. In silico homology modeling of mutant proteins was also performed using available crystallographic data of related nuclear receptors. Results: The mutants p.W234S, p.A256V, p.A256E, p.L263P, p.R309G, p.R311Q, p.R334G, p.L336P, p.L353V, p.R385P and p.M407K, all located in the LBD, showed impaired receptor dimerization at various degrees. Impaired repressor dimerization as assessed by BRET2 assays did not always correlate with impaired repressor function of NR2E3 as assessed by cell-based reporter assays. There were minor differences of transcriptional activity of mutant proteins on mouse S-opsin (opn1sw), mouse cone arrestin (arr3) and human cone arrestin, suggesting that the effect of LBD mutations was independent of the promoter context. Conclusions: Mutational analysis and homology modeling allowed the characterization of potential oligomerization interfaces of the NR2E3 LBD. Additionally, mutations in NR2E3 LBD may cause recessive retinal degenerations by different molecular mechanisms.

Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPARgamma and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPARgamma(1/)- and RXRalpha(1/)- mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPARgamma heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPARgamma agonists. TNBS-induced colitis was significantly reduced by the administration of both PPARgamma and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor alpha and interleukin 1beta mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor kappaB DNA binding activity, c-Jun NH(2)-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPARgamma and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPARgamma heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPARgamma ligands might hold promise in the clinic due to their synergistic effects.

T cell receptor specificity is critical for the development of epidermal gammadelta T cells.

A particular feature of gammadelta T cell biology is that cells expressing T cell receptor (TCR) using specific Vgamma/Vdelta segments are localized in distinct epithelial sites, e.g., in mouse epidermis nearly all gammadelta T cells express Vgamma3/Vdelta1. These cells, referred to as dendritic epidermal T cells (DETC) originate from fetal Vgamma3+ thymocytes. The role of gammadelta TCR specificity in DETC's migration/localization to the skin has remained controversial. To address this issue we have generated transgenic (Tg) mice expressing a TCR delta chain (Vdelta6.3-Ddelta1-Ddelta2-Jdelta1-Cdelta), which can pair with Vgamma3 in fetal thymocytes but is not normally expressed by DETC. In wild-type (wt) Vdelta6.3Tg mice DETC were present and virtually all of them express Vdelta6.3. However, DETC were absent in TCR-delta(-/-) Vdelta6.3Tg mice, despite the fact that Vdelta6.3Tg gammadelta T cells were present in normal numbers in other lymphoid and nonlymphoid tissues. In wt Vdelta6.3Tg mice, a high proportion of in-frame Vdelta1 transcripts were found in DETC, suggesting that the expression of an endogenous TCR-delta (most probably Vdelta1) was required for the development of Vdelta6.3+ epidermal gammadelta T cells. Collectively our data demonstrate that TCR specificity is essential for the development of gammadelta T cells in the epidermis. Moreover, they show that the TCR-delta locus is not allelically excluded.

Constitutive activity and inverse agonism at the α(₁a) and α(₁b) adrenergic receptor subtypes.

The α(1b)-adrenergic receptor (AR) was, after rhodopsin, the first G protein-coupled receptor (GPCR) in which point mutations were shown to trigger constitutive (agonist-independent) activity. Constitutively activating mutations have been found in other AR subtypes as well as in several GPCRs. This chapter briefly summarizes the main findings on constitutively active mutants of the α(1a)- and α(1b)-AR subtypes and the methods used to predict activating mutations, to measure constitutive activity of Gq-coupled receptors and to investigate inverse agonism. In addition, it highlights the implications of studies on constitutively active AR mutants on elucidating the molecular mechanisms of receptor activation and drug action.

Immune response to mouse mammary tumor virus in mice lacking the alpha/beta interferon or the gamma interferon receptor.

Mouse mammary tumor virus (MMTV) is a retrovirus which induces a strong immune response and a dramatic increase in the number of infected cells through the expression of a superantigen (SAg). Many cytokines are likely to be involved in the interaction between MMTV and the immune system. In particular, alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) exert many antiviral and immunomodulatory activities and play a critical role in other viral infections. In this study, we have investigated the importance of interferons during MMTV infection by using mice with a disrupted IFN-alpha/beta or IFN-gamma receptor gene. We found that the SAg response to MMTV was not modified in IFN-alpha/betaR(0/0) and IFN-gammaR(0/0) mice. This was true both for the early expansion of B and T cells induced by the SAg and for the deletion of SAg-reactive cells at later stages of the infection. In addition, no increase in the amount of proviral DNA was detected in tissues of IFN-alpha/betaR(0/0) and IFN-gammaR(0/0) mice, suggesting that interferons are not essential antiviral defense mechanisms during MMTV infection. In contrast, IFN-gammaR(0/0) mice had increased amounts of IL-4 mRNA and an altered usage of immunoglobulin isotypes with a reduced frequency of IgG2a- and IgG3-producing cells. This was associated with lower titers of virus-specific antibodies in serum early after infection, although efficient titers were reached later.

Delayed mortality and attenuated thrombocytopenia associated with severe malaria in urokinase- and urokinase receptor-deficient mice.

We explored the role of urokinase and tissue-type plasminogen activators (uPA and tPA), as well as the uPA receptor (uPAR; CD87) in mouse severe malaria (SM), using genetically deficient (-/-) mice. The mortality resulting from Plasmodium berghei ANKA infection was delayed in uPA(-/-) and uPAR(-/-) mice but was similar to that of the wild type (+/+) in tPA(-/-) mice. Parasitemia levels were similar in uPA(-/-), uPAR(-/-), and +/+ mice. Production of tumor necrosis factor, as judged from the plasma level and the mRNA levels in brain and lung, was markedly increased by infection in both +/+ and uPAR(-/-) mice. Breakdown of the blood-brain barrier, as evidenced by the leakage of Evans Blue, was similar in +/+ and uPAR(-/-) mice. SM was associated with a profound thrombocytopenia, which was attenuated in uPA(-/-) and uPAR(-/-) mice. Administration of aprotinin, a plasmin antagonist, also delayed mortality and attenuated thrombocytopenia. Platelet trapping in cerebral venules or alveolar capillaries was evident in +/+ mice but absent in uPAR(-/-) mice. In contrast, macrophage sequestration in cerebral venules or alveolar capillaries was evident in both +/+ and uPAR(-/-) mice. Polymorphonuclear leukocyte sequestration in alveolar capillaries was similar in +/+ and uPAR(-/-) mice. These results demonstrate that the uPAR deficiency attenuates the severity of SM, probably by its important role in platelet kinetics and trapping. These results therefore suggest that platelet sequestration contributes to the pathogenesis of SM.

Impacte dels nivells de Ciclosporina en el desenvolupament de malaltia de l’empelt contra el receptor aguda en el trasplantament hemopoètic amb acondicionament d’intensitat reduïda de germà HLA idèntic.

Uns nivells correctes en sèrum de Ciclosporina A les primeres setmanes desprès d’ un trasplantament al•logènic amb acondicionament mieloablatiu, disminueixen l’aparició de malaltia de l’empelt contra el receptor aguda (MECRa). El seu impacte en el trasplantament d’intensitat reduïda (alo-TIR) encara no és conegut. En aquest treball retrospectiu s’analitzen les dades d’una cohort molt homogènia de pacients del nostre centre, estudiant-se les seves característiques clíniques i la relació entre els nivells de CsA i l’aparició de MECRa moderada o severa, en la fase precoç de l’alo-TIR.

New insights into the regulatory mechanisms of the NALP3 inflammasome

Résumé : Les vertébrés ont recours au système immunitaire inné et adaptatif pour combattre les pathogènes. La découverte des récepteurs Toll, il y a dix ans, a fortement augmenté l'intérêt porté à l'immunité innée. Depuis lors, des récepteurs intracellulaires tels que les membres de la famille RIG-like helicase (RLHs) et NOD-like receptor (NLRs) ont été décrits pour leur rôle dans la détection des pathogènes. L'interleukine-1 beta (IL-1β) est une cytokine pro-inflammatoire qui est synthétisée sous forme de précurseur, la proIL-1β. La proIL-1β requiert d'être clivée par la caspase-1 pour devenir active. La caspase-1 est elle-même activée par un complexe appelé inflammasome qui peut être formé par divers membres de la famille NLR. Plusieurs inflammasomes ont été décrits tels que le NALP3 inflammasome ou l'IPAF inflammasome. Dans cette étude nous avons identifié la co-chaperone SGT1 et la chaperone HSP90 comme partenaires d'interaction de NALP3. Ces deux protéines sont bien connues chez les plantes pour leurs rôles dans la régulation des gènes de résistance (gène R) qui sont structurellement apparentés à la famille NLR. Nous avons pu montrer que SGT1 et HSP90 jouent un rôle similaire dans la régulation de NALP3 et des protéines R. En effet, nous avons démontré que les deux protéines sont nécessaires pour l'activité du NALP3 inflammasome. De plus, la HSP90 est également requise pour la stabilité de NALP3. En se basant sur ces observations, nous avons proposé un modèle dans lequel SGT1 et HSP90 maintiennent NALP3 inactif mais prêt à percevoir un ligand activateur qui initierait la cascade inflammatoire. Nous avons également montré une interaction entre SGT1 et HSP90 avec plusieurs NLRs. Cette observation suggère qu'un mécanisme similaire pourrait être impliqué dans la régulation des membres de la famille des NLRs. Ces dernières années, plusieurs PAMPs mais également des DAMPs ont été identifiés comme activateurs du NALP3 inflammasome. Dans la seconde partie de cette étude, nous avons identifié la réponse au stress du réticulum endoplasmique (RE) comme nouvel activateur du NALP3 inflammasome. Cette réponse est initiée lors de l'accumulation dans le réticulum endoplasmique de protéines ayant une mauvaise conformation ce qui conduit, en autre, à l'arrêt de la synthèse de nouvelles protéines ainsi qu'une augmentation de la dégradation des protéines. Les mécanismes par lesquels la réponse du réticulum endoplasmique induit l'activation du NALP3 inflammasome doivent encore être déterminés. Summary : Vertebrates rely on the adaptive and the innate immune systems to fight pathogens. Awarness of the importance of the innate system increased with the identification of Toll-like receptors a decade ago. Since then, intracellular receptors such as the RIG-like helicase (RLH) and the NOD-like receptor (NLR) families have been described for their role in the recognition of microbes. Interleukin- 1ß (IL-1ß) is a key mediator of inflammation. This proinflammatory cytokine is synthesised as an inactive precursor that requires processing by caspase-1 to become active. Caspase-1 is, itself, activated in a complex termed the inflammasome that can be formed by members of the NLR family. Various inflammasome complexes have been described such as the IPAF and the NALP3 inflammasome. In this study, we have identified the co-chaperone SGT1 and the chaperone HSP90 as interacting partners of NALP3. SGT1 and HSP90 are both known for their role in the activity of plant resistance proteins (R proteins) which are structurally related to the NLR family. We have shown that HSP90 and SGT1 play a similar role in the regulation of NALP3 and in the regulation of plant R proteins. Indeed, we demonstrated that both HSP90 and SGT1 are essential for the activity of the NALP3 inflammasome complex. In addition, HSP90 is required for the stability of NALP3. Based on these observations, we have proposed a model in which SGT1 and HSP90 maintain NALP3 in an inactive but signaling-competent state, ready to receive an activating ligand that induces the inflammatory cascade. An interaction between several NLR members, SGTI and HSP90 was also shown, suggesting that similar mechanisms could be involved in the regulation of other NLRs. Several pathogen-associated molecular patterns (PAMPs) but also danger associated molecular patterns (DAMPs) have been identified as NALP3 activators. In the second part of this study, we have identified the ER stress response as a new NALP3 activator. The ER stress response is activated upon the accumulation of unfolded protein in the endoplasmic reticulum and results in a block in protein synthesis and increased protein degradation. The mechanisms of ER stress-mediated NALP3 activation remain to be determined.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

Sawhorse-type diruthenium tetracarbonyl complexes containing porphyrin-derived ligands as highly selective photosensitizers for female reproductive cancer cells.

Diruthenium tetracarbonyl complexes of the type [Ru2(CO)4(l2-g2-O2CR)2L2] containing a Ru-Ru backbone with four equatorial carbonyl ligands, two carboxylato bridges, and two axial two-electron ligands in a sawhorse-like geometry have been synthesized with porphyrin-derived substituents in the axial ligands [1: R is CH3, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin], in the bridging carboxylato ligands [2: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is PPh3; 3: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 1,3,5-triaza-7-phosphatricyclo [3.3.1.1]decane], or in both positions [4: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin]. Compounds 1-3 were assessed on different types of human cancer cells and normal cells. Their uptake by cells was quantified by fluorescence and checked by fluorescence microscopy. These compounds were taken up by human HeLa cervix and A2780 and Ovcar ovarian carcinoma cells but not by normal cells and other cancer cell lines (A549 pulmonary, Me300 melanoma, PC3 and LnCap prostate, KB head and neck, MDAMB231 and MCF7 breast, or HT29 colon cancer cells). The compounds demonstrated no cytotoxicity in the absence of laser irradiation but exhibited good phototoxicities in HeLa and A2780 cells when exposed to laser light at 652 nm, displaying an LD50 between 1.5 and 6.5 J/cm2 in these two cell lines and more than 15 J/cm2 for the others. Thus, these types of porphyric compound present specificity for cancer cell lines of the female reproductive system and not for normal cells; thus being promising new organometallic photosensitizers.

Comparison between computational alanine scanning and per-residue binding free energy decomposition for protein-protein association using MM-GBSA: application to the TCR-p-MHC complex.

Recognition by the T-cell receptor (TCR) of immunogenic peptides (p) presented by Class I major histocompatibility complexes (MHC) is the key event in the immune response against virus-infected cells or tumor cells. A study of the 2C TCR/SIYR/H-2K(b) system using a computational alanine scanning and a much faster binding free energy decomposition based on the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method is presented. The results show that the TCR-p-MHC binding free energy decomposition using this approach and including entropic terms provides a detailed and reliable description of the interactions between the molecules at an atomistic level. Comparison of the decomposition results with experimentally determined activity differences for alanine mutants yields a correlation of 0.67 when the entropy is neglected and 0.72 when the entropy is taken into account. Similarly, comparison of experimental activities with variations in binding free energies determined by computational alanine scanning yields correlations of 0.72 and 0.74 when the entropy is neglected or taken into account, respectively. Some key interactions for the TCR-p-MHC binding are analyzed and some possible side chains replacements are proposed in the context of TCR protein engineering. In addition, a comparison of the two theoretical approaches for estimating the role of each side chain in the complexation is given, and a new ad hoc approach to decompose the vibrational entropy term into atomic contributions, the linear decomposition of the vibrational entropy (LDVE), is introduced. The latter allows the rapid calculation of the entropic contribution of interesting side chains to the binding. This new method is based on the idea that the most important contributions to the vibrational entropy of a molecule originate from residues that contribute most to the vibrational amplitude of the normal modes. The LDVE approach is shown to provide results very similar to those of the exact but highly computationally demanding method.

CD8 beta increases CD8 coreceptor function and participation in TCR-ligand binding.

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.