ppGpp controlled by the Gac/Rsm regulatory pathway sustains biocontrol activity in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.

Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins.

Adrenoceptors are prototypic members of the superfamily of seven transmembrane domain, G protein-coupled receptors. Study of the properties of several mutationally activated adrenoceptors is deepening understanding of the normal functioning of this ubiquitous class of receptors. The new findings suggest an expansion of the classical ternary complex model of receptor action to include an explicit isomerization of the receptors from an inactive to an active state which couples to the G protein ('allosteric ternary complex model'). This isomerization involves conformational changes which may occur spontaneously, or be induced by agonists or appropriate mutations which abrogate the normal 'constraining' function of the receptor, allowing it to 'relax' into the active conformation. Robert Lefkowitz and colleagues discuss the physiological and pathophysiological implications of these new insights into regulation of receptor activity.

The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells.

Susceptibility and development of Th2 cells in BALB/c mice infected with Leishmania major result from early IL-4 production by Vbeta4Valpha8 CD4+ T cells in response to the Leishmania homolog of mammalian RACK1 Ag. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. We further showed that transfer of 10(7) BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 10(7) control BALB/c spleen cells were resistant to infection with L. major and developed a Th1 response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.

The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.

A key regulatory role of the transcription factor NFATc2 in bronchial adenocarcinoma via CD8+ T lymphocytes.

The Ca(2+)-regulated calcineurin/nuclear factor of activated T cells (NFAT) cascade controls alternative pathways of T-cell activation and peripheral tolerance. Here, we describe reduction of NFATc2 mRNA expression in the lungs of patients with bronchial adenocarcinoma. In a murine model of bronchoalveolar adenocarcinoma, mice lacking NFATc2 developed more and larger solid tumors than wild-type littermates. The extent of central tumor necrosis was decreased in the tumors in NFATc2((-/-)) mice, and this finding was associated with reduced tumor necrosis factor-alpha and interleukin-2 (IL-2) production by CD8(+) T cells. Adoptive transfer of CD8(+) T cells of NFATc2((-/-)) mice induced transforming growth factor-beta(1) in the airways of recipient mice, thus supporting CD4(+)CD25(+)Foxp-3(+)glucocorticoid-induced tumor necrosis factor receptor (GITR)(+) regulatory T (T(reg)) cell survival. Finally, engagement of GITR in NFATc2((-/-)) mice induced IFN-gamma levels in the airways, reversed the suppression by T(reg) cells, and costimulated effector CD4(+)CD25(+) (IL-2Ralpha) and memory CD4(+)CD127(+) (IL-7Ralpha) T cells, resulting in abrogation of carcinoma progression. Agonistic signaling through GITR, in the absence of NFATc2, thus emerges as a novel possible strategy for the treatment of human bronchial adenocarcinoma in the absence of NFATc2 by enhancing IL-2Ralpha(+) effector and IL-7Ralpha(+) memory-expressing T cells.

Influence of CD4+/CD25+ regulatory T cells on atherogenesis in patients with end-stage kidney disease.

Atherosclerosis, which is influenced by both traditional and nontraditional cardiovascular risk factors and has been characterized as an inflammatory process, is considered to be the main cause of the elevated cardiovascular risk associated with chronic kidney disease. The inflammatory component of atherosclerosis can be separated into an innate immune response involving monocytes and macrophages that respond to the excessive uptake of lipoproteins and an adaptive immune response that involves antigen-specific T cells. Concurrent with the influx of immune cells to the site of atherosclerotic lesion, the role of the adaptive immune response gradually increases. One of those cells are represented by the CD4+/CD25+ Tregs, which play indispensable roles in the maintenance of natural self-tolerance and negative control of pathological, as well as physiological, immune responses. Altered self-antigens such as oxidized LDLs may induce the development of CD4+/CD25+ Tregs with atheroprotective properties. However, atherosclerosis may be promoted by an imbalance between regulatory and pathogenic immunity that may be represented by the low expression of the forkhead box transcription factor (Foxp3) in CD4+/CD25+ Tregs. Such a defect may break immunologic tolerance and alter both specific and bystander immune suppression, leading to exacerbation of plaque development. Patients with end-stage kidney disease (ESKD) display a cellular immune dysfunction and accelerated atherosclerosis. Uremic solutes that accumulate during ESKD may be involved in these processes. In patients with ESKD and especially in those that are chronically hemodialyzed, oxidative stress induced by oxidized LDLs may increase CD4+/CD25+ Treg sensitivity to Fas-mediated apoptosis as a consequence of specific dysregulation of IL-2 expression. This review will focus on the current state of knowledge regarding the influence of CD4+/CD25+ Tregs on atherogenesis in patients with ESKD, and the potential effect of statins on the circulating number and the functional properties of these cells.

A regulatory role for the cohesin loader NIPBL in nonhomologous end joining during immunoglobulin class switch recombination.

DNA double strand breaks (DSBs) are mainly repaired via homologous recombination (HR) or nonhomologous end joining (NHEJ). These breaks pose severe threats to genome integrity but can also be necessary intermediates of normal cellular processes such as immunoglobulin class switch recombination (CSR). During CSR, DSBs are produced in the G1 phase of the cell cycle and are repaired by the classical NHEJ machinery. By studying B lymphocytes derived from patients with Cornelia de Lange Syndrome, we observed a strong correlation between heterozygous loss-of-function mutations in the gene encoding the cohesin loading protein NIPBL and a shift toward the use of an alternative, microhomology-based end joining during CSR. Furthermore, the early recruitment of 53BP1 to DSBs was reduced in the NIPBL-deficient patient cells. Association of NIPBL deficiency and impaired NHEJ was also observed in a plasmid-based end-joining assay and a yeast model system. Our results suggest that NIPBL plays an important and evolutionarily conserved role in NHEJ, in addition to its canonical function in sister chromatid cohesion and its recently suggested function in HR.

CD4+CD25+Foxp3+ regulatory T cells: from basic research to potential therapeutic use.

Regulatory T cells control immune responses to self- and foreign-antigens and play a major role in maintaining the balance between immunity and tolerance. This article reviews recent key developments in the field of CD4+CD25+Foxp3+ regulatory T (TREG) cells. It presents their characteristics and describes their range of activity and mechanisms of action. Some models of diseases triggered by the imbalance between TREG cells and effector pathogenic T cells are described and their potential therapeutic applications in humans are outlined.

Cognate microRNA-Gene Regulatory Networks Mediating Glioblastoma Oncogenic Properties

Malignant gliomas, including the most common and fatal form glioblastoma (GBM, WHO grade IV astrocytoma), remain a challenge to treat. In the United States and Europe, more than 30,000 patients per year are newly diagnosed with GBM. Despite ongoing trials, the best currently available multimodal treatment approaches include surgical resection followed by concomitant and adjuvant radiation (RT) and temozolomide (TMZ) therapy, resulting in a low median overall survival (OS) rate ranging from 12.2 - 15.9 months. The important role of genetic and epigenetic changes in DNA, RNA, and protein alteration as well as epigenetic changes secondary to the tumor microenvironment and outside selection pressure (therapeutic interventions), are increasingly being recognized. In GBM treatment, the focus is shifting toward a more patient-centered (personalized) therapy. In this regard, in particular, microRNAs are being increasingly studied. MicroRNAs are non¬protein coding small RNAs that serve as negative gene regulators by binding to a specific sequence in the promoter region of a target gene, thus regulating gene expression. A single microRNA potentially targets hundreds of genes; thus, microRNAs and their cognate target genes have important roles as tumor suppressors and oncogenes as well as regulators of various cancer- specific cellular features, such as proliferation, apoptosis, invasion, and metastasis. The identification of distinct microRNA-gene regulatory networks in GBM patients can be expected to provide novel therapeutic insights by identifying candidate patients for targeted therapies. To this end, in this work we identified and validated clinically relevant and meaningful novel gene- microRNA regulatory networks that correlated with MR tumor phenotypes, histopathology, and patient survival and response rates to therapy. - Le traitement des gliomes malins, y compris sous leur forme la plus commune et meurtrière, le glioblastome (GBM, ou astrocytome de grade IV selon l'OMS), demeure à ce jour un défi. Aux États-Unis et en Europe, un nouveau diagnostic de GBM est prononcé dans plus de 30Ό00 cas par an. En dépit de tests en cours, les meilleures approches thérapeutiques combinées actuellement disponibles comprennent la résection chirurgicale de la tumeur, suivie d'une radiothérapie adjuvante ainsi que d'un traitement au temozolomide (RT/TMZ), thérapies dont résulte une médiane de survie globale basse (overall survival, OS), comprise entre 12.2 et 15.9 mois. On reconnaît de plus en plus le rôle majeur de l'ADN, de l'ARN et de l'altération des protéines ainsi que des modifications épigénétiques, secondaires par rapport au microenvironnement de la tumeur et à la pression de sélection extérieure (les interventions thérapeutiques). Dans le traitement du GBM, le centre d'intérêt se déplace vers une thérapie centrée sur le cas individuel du patient. Dans ce but, en particulier les microARN sont de plus en plus analysés. Les microARN sont de petits ARN non-codants (les protéines) qui servent de régulateurs négatifs de gènes en s'attachant à une séquence spécifique dans la région promotrice d'un gène-cible, régulant ainsi l'expression du gène. Un seul microARN cible potentiellement des centaines de gènes; on a ainsi découvert que les microARN et leurs gènes-cibles apparentés ont une fonction importante en tant que suppresseurs de tumeurs et d'oncogènes, ainsi que comme régulateurs de diverses caractéristiques cellulaires spécifiques du cancer, comme la prolifération, l'apoptose, l'invasion et la métastase. On peut s'attendre à ce que l'identification de réseaux microARN régulateurs de gènes, distincts selon les patients de GBM, fournisse une approche thérapeutique inédite par la détermination des patients susceptibles de réagir favorablement à des thérapies ciblées.

Valproate treatment of human cord blood CD4-positive effector T cells confers on them the molecular profile (microRNA signature and FOXP3 expression) of natural regulatory CD4-positive cells through inhibition of histone deacetylase.

Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.

Streptozotocin induces a shift towards T regulatory cell responses in vivo

Objectives: Streptozotocin (STZ) induced diabetes is currently the most commonly used animalmodel for islet transplantation.However, STZtreatment and the ensuing hyperglycemia were both shown to affect the immune response, including an apparent induction of lymphopenia. The aim of this study was to evaluate the respective effect of STZ and hyperglycemia on the immune system in STZ induced diabetic C57BL/6 mice. Methods: Phenotypes and levels of T and B cells were analyzed by flow cytometry in blood and spleen over time. The effect of hyperglycemia was further characterized by insulin replacement, islet transplantation and by using Rip (rat insulin promoter) DTR (dipheteria tocin receptor) transgenic mice. Results: STZ but not hyperglycemia was toxic for splenocytes in vitro, whereas hyperglycemia correlated with diabetes associated blood and spleen lymphopenia in vivo. Moreover, independently of hyperglycemia, STZ lead to a relative increase of T regulatory cells which retained their suppressive capacity in vitro. Conclusion: These data suggest thatSTZand the ensuing acute hyperglycemia have major direct and indirect effects on immune homeostasis. Thus, high caution needs to be exercised in the interpretation of the results of tolerance induction and/or immunosuppressive protocols in STZ-induced diabetes and islet transplantation models.

Differential effects of immunosuppressive drugs on effector and regulatory T cells.

Purpose: Current experimental data suggest that CD4+CD25+Foxp3+regulatory T cells (Tregs) based immunotherapy would be of greatinterest to promote donor-specific immune tolerance in transplantation(Tx). Whether and how adoptive transfer of Tregs could be bestcombined with current immunosuppressive regimens in clinicalsettings remains to be defined. Using an experimental Tx model,we had previously shown that the transfer of antigen-specific Tregspromoted long-term skin allograft acceptance in lymphopenic mice,in the absence of any immunosuppressive drug. However, allograftsurvival was only slightly prolonged when Tregs were transferredalone into non-lymphopenic mice, suggesting that in more stringentconditions such as in clinical settings adjuvant therapies may beneeded to effectively control alloreactive T cells (Teff).Methods and Materials: Here we have investigated the effects ofvarious immunosuppressive drugs on the survival, proliferation andeffector function of Teff and Tregs in response to alloantigens in in vitroassays and in our in vivo Tx model.Results: Teff proliferation was inhibited in a dose-dependant mannerby rapamycin and cyclosporine A, while anti-CD154 only marginallyaffected Teff proliferation and survival in vitro. Rapamycin promotedapoptosis of Teff as compared to Tregs that were more resistant underthe same culture conditions. In vivo, the transfer of donor-specificTregs could be advantageously combined with rapamycin andanti-CD154 to significantly prolong MHC-mismatched skin allograftsurvival in non-lymphopenic recipients.Conclusion: Taken together, our data indicate thatimmunosuppressive drugs differentially target T-cell subsets and couldpromote Tregs expansion and/or function while controlling the Teff pool.

Spatio-temporal sequence of cross-regulatory events in root meristem growth.

A central question in developmental biology is how multicellular organisms coordinate cell division and differentiation to determine organ size. In Arabidopsis roots, this balance is controlled by cytokinin-induced expression of SHORT HYPOCOTYL 2 (SHY2) in the so-called transition zone of the meristem, where SHY2 negatively regulates auxin response factors (ARFs) by protein-protein interaction. The resulting down-regulation of PIN-FORMED (PIN) auxin efflux carriers is considered the key event in promoting differentiation of meristematic cells. Here we show that this regulation involves additional, intermediary factors and is spatio-temporally constrained. We found that the described cytokinin-auxin crosstalk antagonizes BREVIS RADIX (BRX) activity in the developing protophloem. BRX is an auxin-responsive target of the prototypical ARF MONOPTEROS (MP), a key promoter of vascular development, and transiently enhances PIN3 expression to promote meristem growth in young roots. At later stages, cytokinin induction of SHY2 in the vascular transition zone restricts BRX expression to down-regulate PIN3 and thus limit meristem growth. Interestingly, proper SHY2 expression requires BRX, which could reflect feedback on the auxin responsiveness of SHY2 because BRX protein can directly interact with MP, likely acting as a cofactor. Thus, cross-regulatory antagonism between BRX and SHY2 could determine ARF activity in the protophloem. Our data suggest a model in which the regulatory interactions favor BRX expression in the early proximal meristem and SHY2 prevails because of supplementary cytokinin induction in the later distal meristem. The complex equilibrium of this regulatory module might represent a universal switch in the transition toward differentiation in various developmental contexts.