The vasodilatory effect of juxta-arteriolar microinjection of endothelinA receptor inhibitor in healthy and acute branch retinal vein occlusion minipig retinas.

Purpose. To investigate the effect of the endothelin(A) receptor inhibitor BQ-123 on the retinal arteriolar vasculature in minipig retinas in normal eyes and eyes with acute branch retinal vein occlusion (BRVO). Methods. Seven healthy eyes of seven minipigs and six eyes of six minipigs with experimental BRVO were evaluated under systemic anesthesia. An intravitreal juxta-arteriolar microinjection of 30 microL BQ-123 0.61 microg/mL (pH 7.4) was performed in all but one eye from each group, into which the physiologic saline vehicle alone was injected. Vessel-diameter changes were measured with a retinal vessel analyzer. Results. In healthy minipig retinas (n = 6), arteriolar diameter (+/-SD) increased 6.19% +/- 3.55% (P < 0.05), 25.98% +/- 2.37% (P < 0.001), 23.65% +/- 1.2% (P < 0.001), and 16.84% +/- 1.95% (P < 0.001), at 1, 5, 10, and 15 minutes, respectively, after BQ-123 microinjection. Two hours after experimental BRVO (n = 5), the retinal arteriolar diameter had decreased (13.07% +/- 5.7%; P < 0.01). One, 5, 10, and 15 minutes after BQ-123 microinjection, retinal arteriolar diameter had increased by 7.14% +/- 3.3% (P < 0.01), 26.74% +/- 7.63% (P < 0.001), 23.67% +/- 6.4% (P < 0.001), and 16.09% +/- 3.41% (P < 0.001), respectively. Vehicle only injection had no vasoactive effect on physiologic or BRVO retinas. Conclusions. A significant increase in retinal arteriolar diameter was demonstrated after juxta-arteriolar BQ-123 microinjection in healthy and in acute BRVO minipig retinas. The results suggest a role for endothelin-1 in maintaining retinal basal arteriolar tone. Reversing the BRVO-related vasoconstriction by juxta-arteriolar BQ-123 microinjection could bring a new perspective to the management of BRVO.

Three-dimensional radiobiological dosimetry of kidneys for treatment planning in peptide receptor radionuclide therapy.

PURPOSE: Peptide receptor radionuclide therapy (PRRT) delivers high absorbed doses to kidneys and may lead to permanent nephropathy. Reliable dosimetry of kidneys is thus critical for safe and effective PRRT. The aim of this work was to assess the feasibility of planning PRRT based on 3D radiobiological dosimetry (3D-RD) in order to optimize both the amount of activity to administer and the fractionation scheme, while limiting the absorbed dose and the biological effective dose (BED) to the renal cortex. METHODS: Planar and SPECT data were available for a patient examined with (111)In-DTPA-octreotide at 0.5 (planar only), 4, 24, and 48 h post-injection. Absorbed dose and BED distributions were calculated for common therapeutic radionuclides, i.e., (111)In, (90)Y and (177)Lu, using the 3D-RD methodology. Dose-volume histograms were computed and mean absorbed doses to kidneys, renal cortices, and medullae were compared with results obtained using the MIRD schema (S-values) with the multiregion kidney dosimetry model. Two different treatment planning approaches based on (1) the fixed absorbed dose to the cortex and (2) the fixed BED to the cortex were then considered to optimize the activity to administer by varying the number of fractions. RESULTS: Mean absorbed doses calculated with 3D-RD were in good agreement with those obtained with S-value-based SPECT dosimetry for (90)Y and (177)Lu. Nevertheless, for (111)In, differences of 14% and 22% were found for the whole kidneys and the cortex, respectively. Moreover, the authors found that planar-based dosimetry systematically underestimates the absorbed dose in comparison with SPECT-based methods, up to 32%. Regarding the 3D-RD-based treatment planning using a fixed BED constraint to the renal cortex, the optimal number of fractions was found to be 3 or 4, depending on the radionuclide administered and the value of the fixed BED. Cumulative activities obtained using the proposed simulated treatment planning are compatible with real activities administered to patients in PRRT. CONCLUSIONS: The 3D-RD treatment planning approach based on the fixed BED was found to be the method of choice for clinical implementation in PRRT by providing realistic activity to administer and number of cycles. While dividing the activity in several cycles is important to reduce renal toxicity, the clinical outcome of fractionated PRRT should be investigated in the future.

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

The transcription factor c-Maf controls touch receptor development and function.

The sense of touch relies on detection of mechanical stimuli by specialized mechanosensory neurons. The scarcity of molecular data has made it difficult to analyze development of mechanoreceptors and to define the basis of their diversity and function. We show that the transcription factor c-Maf/c-MAF is crucial for mechanosensory function in mice and humans. The development and function of several rapidly adapting mechanoreceptor types are disrupted in c-Maf mutant mice. In particular, Pacinian corpuscles, a type of mechanoreceptor specialized to detect high-frequency vibrations, are severely atrophied. In line with this, sensitivity to high-frequency vibration is reduced in humans carrying a dominant mutation in the c-MAF gene. Thus, our work identifies a key transcription factor specifying development and function of mechanoreceptors and their end organs.

Nuclear triiodothyronine receptor expression is regulated by axon-Schwann cell contact.

Thyroid hormones, which play an important role in the development and regeneration of the nervous system, require the presence of specific nuclear T3 receptors (NT3R). In this study we provide evidence that NT3R expression by Schwann cells was up-regulated in response to a loss of axonal contact in vitro and in vivo. In dorsal root ganglia explant cultures, Schwann cells which accompanied axons (nerve fibres) were devoid of NT3R. When Schwann cells were orphaned from axon contact by axon transection, all the nuclei of these cells displayed NT3R immunoreactivity. Similar results were obtained in situ; in adult rat sciatic nerve, Schwann cells which ensheathed healthy axons never expressed NT3R immunoreactivity. After sciatic nerve transection in vivo the nuclei of Schwann cells deprived of axonal contact displayed a clear NT3R immunoreaction.

Xenopus peroxisome proliferator activated receptors: genomic organization, response element recognition, heterodimer formation with retinoid X receptor and activation by fatty acids.

Peroxisome proliferator activated receptors are ligand activated transcription factors belonging to the nuclear hormone receptor superfamily. Three cDNAs encoding such receptors have been isolated from Xenopus laevis (xPPAR alpha, beta, and gamma). Furthermore, the gene coding for xPPAR beta has been cloned, thus being the first member of this subfamily whose genomic organization has been solved. Functionally, xPPAR alpha as well as its mouse and rat homologs are thought to play an important role in lipid metabolism due to their ability to activate transcription of a reporter gene through the promoter of the acyl-CoA oxidase (ACO) gene. ACO catalyzes the rate limiting step in the peroxisomal beta-oxidation of fatty acids. Activation is achieved by the binding of xPPAR alpha on a regulatory element (DR1) found in the promoter region of this gene, xPPAR beta and gamma are also able to recognize the same type of element and are, as PPAR alpha, able to form heterodimers with retinoid X receptor. All three xPPARs appear to be activated by synthetic peroxisome proliferators as well as by naturally occurring fatty acids, suggesting that a common mode of action exists for all the members of this subfamily of nuclear hormone receptors.

Ectodysplasin A (EDA)-EDA receptor signalling and its pharmacological modulation

L'ectodysplasine Al (EDA1 ou EDA), un ligand de la famille du TNF, et son récepteur EDAR favorisent le développement des poils, des dents et de plusieurs types de glandes. Chez l'humain, une déficience en EDA cause une dysplasie ectodermique liée à l'X, caractérisée par la genèse défectueuse des phanères. Les souris Tabby, déficientes en Eda, présentent des symptômes similaires. Nous démontrons que les souris Tabby sont en moyenne 7% plus légères que les contrôles au moment du sevrage. Ce phénotype ne dépend pas du génotype des petits, mais exclusivement de celui de la mère, suggérant que l'absence d'EDA perturbe la fonction mammaire. La glande mammaire se développe en plusieurs étapes, principalement à la puberté et pendant la grossesse. Nous avons généré des anticorps pour activer ou inhiber la signalisation d'EDAR. Les anticorps agonistes corrigent le développement de souris ou de chiens déficients en EDA, alors que les antagonistes provoquent une dysplasie ectodermique chez les souris saines. L'exposition répétée de souris Tabby aux anticorps agonistes après le sevrage accroît la taille et la fonction des glandes sébacées, démonstration pharmacologique qu'EDA contrôle l'homéostasie de la glande sébacée adulte. Ces outils seront utiles pour étudier la fonction d'EDA aux diverses étapes du développement de la glande mammaire. Fc-EDAl, un stimulateur d'EDAR, est en phase d'évaluation clinique. Nous avons montré que les structures dépendantes d'EDA qui se forment à différentes étapes du développement répondent à l'action du Fc-EDAl dans des fenêtres temporelles étroites ou larges. De plus, certaines structures peuvent être induites plusieurs jours après le début naturel de leur formation. Alors que la plupart des structures se forment suite à un seul jour d'activation d'EDAR, d'autre demandent un temps de stimulation plus long. La formation des dents est régulée par des signaux activateurs et inhibiteurs. Une forte stimulation d'EDAR spécifiquement appliquée aux deux premières molaires induit des signaux négatifs qui avortent la formation de la troisième molaire, alors qu'une forte stimulation donnée à la troisième molaire la rend hypertrophique tout en induisant parfois une quatrième molaire jamais observée chez les souris de type sauvage ou Tabby. EDA est donc un activateur important de la formation dentaire. Pris dans leur ensemble, ces résultats ont des implications pour la thérapie des dysplasies ectodermiques. - The TNF family ligand Ectodysplasin Al (EDA1 or EDA) and its receptor ED AR regulate embryonic development of hair, teeth and several types of glands. In humans, EDA mutations cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition characterized by defective development of skin appendages. £da-deficient (Tabby) mice suffer from similar defects. We observed that Tabby pups at weaning were on average 7% smaller than WT controls, a phenotype that was curiously not linked to the genotype of pups, but to that of mothers, suggesting decreased mammary gland function in the absence of EDA. Mammary glands develop in several steps, most of which are post-natal. We generated monoclonal antibodies to block or activate EDAR signaling. Agonist antibodies rescued developmental defects when administered timely in £cfo-deficient mice and dogs, whereas blocking antibodies induced ectodermal dysplasia in WT mice. Agonist antibodies administered after weaning in £da-deficient mice for several months markedly increased both size and function of sebaceous glands, providing the first demonstration that pharmacological activation of the EDAR pathway in adults can correct important aspects of the dry skin phenotype. This also highlights a role for EDA1 in the homeostasis of adult sebaceous glands. These tools will be useful to study the function of EDA 1 at different stages of mammary gland development. Another EDAR agonist, Fc-EDAl, is currently evaluated in clinical trials. We found that EDA 1-dependent structures forming at different time points during development can respond to Fc-EDAl during time response windows that are narrow or wide. Also, some structures can be triggered up to several days after their normal time of induction. While most structures could be rescued by a single day of EDAR signaling, others required longer exposure times to form. Tooth formation is regulated by activating and inhibitory signals that impact one on the other. When strong EDAR signals were specifically given to the first two molars, overwhelming inhibitory signals completely inhibited formation of the third molar. In contrast, strong signals specifically given to the third molar induced hypertrophy of the later with occasional appearance of a fourth molar never observed in WT or £da-deficient mice. This clearly positions EDA as an important activating signal in tooth formation. Taken together, these results have implications for the therapy of ectodermal dysplasias.

Optimal T-cell receptor affinity for inducing autoimmunity.

T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity.

A soluble form of B cell maturation antigen, a receptor for the tumor necrosis factor family member APRIL, inhibits tumor cell growth.

A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.

Interleukins (IL)-1 and IL-2 control IL-2 receptor alpha and beta expression in immature thymocytes.

Functional high-affinity interleukin-2 receptors (IL-2R) contain three transmembrane proteins, IL-2R alpha, beta and gamma. We have investigated the expression of IL-2R alpha and beta genes in immature mouse thymocytes. Previous work has shown that during differentiation these cells transiently express IL-2R alpha on their surface. Stimulation of IL-2R alpha+ and IL-2R alpha- immature thymocytes with phorbol 12-myristate 13-acetate and calcium ionophore induces synthesis of IL-2R alpha and IL-2R beta mRNA. Most of this response depends on autocrine stimulation by IL-2. IL-1 synergizes with IL-2 to induce a 120-fold increase in IL-2R alpha mRNA and a 14-fold increase in IL-2R beta mRNA levels. A large proportion of the stimulated cells contains both transcripts. These interleukins do not induce any differentiation to more mature phenotypes. Collectively, these results show that IL-2 plays a major role in the regulation of IL-2R expression in normal immature thymocyte. We suggest that this response to interleukins may be part of a homeostatic mechanism to increase the production of immature thymocytes during stress.

The caspase 8 inhibitor c-FLIP(L) modulates T-cell receptor-induced proliferation but not activation-induced cell death of lymphocytes.

The caspase 8 inhibitor c-FLIP(L) can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIP(L) in the T-cell compartment (c-FLIP(L) Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIP(L) Tg mice. In contrast, activation-induced cell death of T cells in c-FLIP(L) Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIP(L) Tg mice differed from Fas-deficient mice by showing no accumulation of B220(+) CD4(-) CD8(-) T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIP(L) Tg mice. Thus, a major role of c-FLIP(L) in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.

Mineralocorticoid Receptor Antagonism in the Treatment of Chronic Central Serous Chorioretinopathy : A Pilot Study.

PURPOSE:: Based on experimental data showing that central serous chorioretinopathy could result from overactivation of mineralocorticoid receptor pathway in choroid vessels, the authors studied eplerenone, a mineralocorticoid receptor antagonist, as a potential treatment for chronic central serous chorioretinopathy. METHODS:: This nonrandomized pilot study included 13 patients with central serous chorioretinopathy of at least 4-month duration, treated with 25 mg/day of oral eplerenone for a week followed by 50 mg/day for 1 or 3 months. The primary outcome measure was the changes in central macular thickness recorded by optical coherence tomography, and the secondary outcomes included changes in foveal subretinal fluid (SRF) measured by OCT, in best-corrected visual acuity (BCVA) and the percentage of eyes achieving complete resolution of subretinal fluid during the treatment period. RESULTS:: Central macular thickness decreased significantly from 352 ± 139 μm at baseline to 246 ± 113 μm and 189 ± 99 μm at 1 and 3 months under eplerenone treatment (P < 0.05 and P < 0.01, respectively). At 3 months, the subretinal fluid significantly decreased compared with baseline subretinal fluid (P < 0.01) and best-corrected visual acuity significantly improved compared with baseline best-corrected visual acuity (P < 0.001). CONCLUSION:: Eplerenone treatment was associated with a significant reduction in central macular thickness, subretinal fluid level, and an improvement in visual acuity. Randomized controlled trials are needed to confirm these encouraging results.

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts.

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.

Antagonistes de l'angiotensine II et cancer: un bilan rassurant [Angiotensin II receptor blockers (ARBs) and cancer: a reassuring balance].

The effects of drugs on new cancer and cancer-related death are a major concern. Recently, a meta-analysis raised the possibility that ARBs might have an adverse impact in this respect. This point of view was highly debated until the publication of two other meta-analyses which did not demonstrate any increased risk of new cancer occurrence as well as of cancer related-death with the use of ARBs in patients with hypertension, heart failure and/or nephropathy. This illustrates that the results of meta-analyses should be interpreted cautiously and critically in order to avoid biased conclusions. Overall the bulk of evidence today indicates that ARBs are not associated with an increased cancer risk.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.