Increased frequency of regulatory T cells and selection of highly potent CD62L+ cells during treatment of human lung transplant recipients with rapamycin.

The currently available immunosuppressive agents applied in human transplantation medicine are highly potent in the protection from acute allograft rejection. However, long-term allograft survival is still poor as these drugs fail to sufficiently prevent chronic allograft rejection. Naturally occurring regulatory T cells have been postulated as the key players to establish long-lasting transplantation tolerance. Thus, the development of immunosuppressive regimens which shift the pathological balance of cytopathic versus regulatory T cells of human allograft recipients towards a protective T-cell composition is a promising approach to overcome limitations of current transplantation medicine. Thirty-three patients that received rapamycin (RPM) or calcineurin inhibitor treatment following lung transplantation were included and their T-cell compartments analysed. Twelve healthy volunteers without history of lung disease served as controls. In this article, we show that treatment of human lung transplant recipients with RPM is associated with an increased frequency of regulatory T cells, as compared with treatment with calcineurin inhibitors or to healthy controls. Moreover, regulatory T cells during treatment with RPM were CD62Lhigh, a phenotype that displayed an enhanced immunosuppressive capacity ex vivo. Our data support the use of RPM in human lung transplant recipients and undertaking of further prospective studies evaluating its impact on allograft and patient survival.

Increased EBV Reactivation in the Cerebrospinal Fluid of Patients with Early Multiple Sclerosis

Epstein-Barr virus (EBV) has been consistently associated with multiple sclerosis (MS), but whether this virus is a trigger of MS remains undetermined. Recently, EBV-infected B cells recognized by activated CD8_ T cells have been detected in the meninges of autopsied MS patients. In addition, a strong EBV-specific CD8_ T cell response in the blood of patients with MS of recent onset was reported. Here, to further explore the putative relationship between MS and EBV, we assessed the EBV-specific cellular and humoral immune responses in the blood and the cerebrospinal fluid (CSF) of patients with early MS or other neurological diseases, separated into inflammatory (IOND) and non-inflammatory (NIOND) groups. The MS non-associated neurotropic herpesvirus cytomegalovirus (CMV) served as a control. Fifty-eight study subjects were enrolled, including 44 patients (13 with early MS (onset of MS less than one year prior to the assay), 15 with IOND and 16 with NIOND) in the immunological arm of the study. The cellular immune response was investigated using a functional CFSE cytotoxic T lymphocyte (CTL) assay performed with short-term cultured EBV- or CMVspecific effector T cells from the CSF and the blood. The humoral immune response specific for these two viruses was also examined in both the blood and the CSF. The recruitment of a given virusspecific antibody in the CSF as compared to the blood was expressed as antibody indexes (AI). We found that, in the CSF of early MS patients, there was an enrichment in EBV-, but not CMV-specific, CD8_ CTL as compared to the CSF of IOND (P_ 0.003) and NIOND patients (P_0.0009), as well as compared to paired blood samples (P_0.005). Additionally, relative viral capsid antigen (VCA)-, but not EBV encoded nuclear antigen 1 (EBNA1)- or CMV-specific, AI were increased in the CSF of early MS as compared to IOND (P_0.002) or NIOND patients (P_0.008) and correlated with the EBVspecific CD8_ CTL responses in the CSF (rs_0.54, P_0.001). Fourteen additional patients were enrolled in the virological arm of the study: using semi-nested PCR, EBV-encoded nuclear RNA1 (EBER1)-a transcript expressed during all stages of EBV infection-was detected in the CSF of 2/4 early MS, but only 1/6 IOND and 0/4 NIOND patients. Altogether, our data suggest that a reactivation of EBV, but not CMV, is taking place in the central nervous system of patients with MS of recent onset. These data significantly strengthen the link between EBV and MS and may indicate a triggering role of EBV in this disease. This work was supported by grants from the Swiss National Foundation and from the Swiss Society for Multiple Sclerosis.

β-Glucan Antigenemia Anticipates Diagnosis of Blood Culture-Negative Intraabdominal Candidiasis.

Rationale: Life-threatening intraabdominal candidiasis (IAC) occurs in 30 to 40% of high-risk surgical intensive care unit (ICU) patients. Although early IAC diagnosis is crucial, blood cultures are negative, and the role of Candida score/colonization indexes is not established. Objectives: The aim of this prospective Fungal Infection Network of Switzerland (FUNGINOS) cohort study was to assess accuracy of 1,3-β-d-glucan (BG) antigenemia for diagnosis of IAC. Methods: Four hundred thirty-four consecutive adults with abdominal surgery or acute pancreatitis and ICU stay 72 hours or longer were screened: 89 (20.5%) at high risk for IAC were studied (68 recurrent gastrointestinal tract perforation, 21 acute necrotizing pancreatitis). Diagnostic accuracy of serum BG (Fungitell), Candida score, and colonization indexes was compared. Measurements and Main Results: Fifty-eight of 89 (65%) patients were colonized by Candida; 29 of 89 (33%) presented IAC (27 of 29 with negative blood cultures). Nine hundred twenty-one sera were analyzed (9/patient): median BG was 253 pg/ml (46-9,557) in IAC versus 99 pg/ml (8-440) in colonization (P < 0.01). Sensitivity and specificity of two consecutive BG measurements greater than or equal to 80 pg/ml were 65 and 78%, respectively. In recurrent gastrointestinal tract perforation it was 75 and 77% versus 90 and 38% (Candida score ≥ 3), 79 and 34% (colonization index ≥ 0.5), and 54 and 63% (corrected colonization index ≥ 0.4), respectively. BG positivity anticipated IAC diagnosis (5 d) and antifungal therapy (6 d). Severe sepsis/septic shock and death occurred in 10 of 11 (91%) and 4 of 11 (36%) patients with BG 400 pg/ml or more versus 5 of 18 (28%, P = 0.002) and 1 of 18 (6%, P = 0.05) with BG measurement less than 400 pg/ml. β-Glucan decreased in IAC responding to therapy and increased in nonresponse. Conclusions: BG antigenemia is superior to Candida score and colonization indexes and anticipates diagnosis of blood culture-negative IAC. This proof-of-concept observation in strictly selected high-risk surgical ICU patients deserves investigation of BG-driven preemptive therapy.

Impaired fast-spiking, suppressed cortical inhibition and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins

Voltage-gated K+ channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to −10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 −/− mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.

Decay of linkage disequilibrium within genes across HGDP-CEPH human samples: most population isolates do not show increased LD

Background: It is well known that the pattern of linkage disequilibrium varies between human populations, with remarkable geographical stratification. Indirect association studies routinely exploit linkage disequilibrium around genes, particularly in isolated populations where it is assumed to be higher. Here, we explore both the amount and the decay of linkage disequilibrium with physical distance along 211 gene regions, most of them related to complex diseases, across 39 HGDP-CEPH population samples, focusing particularly on the populations defined as isolates. Within each gene region and population we use r2 between all possible single nucleotide polymorphism (SNP) pairs as a measure of linkage disequilibrium and focus on the proportion of SNP pairs with r2 greater than 0.8.Results: Although the average r2 was found to be significantly different both between and within continental regions, a much higher proportion of r2 variance could be attributed to differences between continental regions (2.8% vs. 0.5%, respectively). Similarly, while the proportion of SNP pairs with r2 > 0.8 was significantly different across continents for all distance classes, it was generally much more homogenous within continents, except in the case of Africa and the Americas. The only isolated populations with consistently higher LD in all distance classes with respect to their continent are the Kalash (Central South Asia) and the Surui (America). Moreover, isolated populations showed only slightly higher proportions of SNP pairs with r2 > 0.8 per gene region than non-isolated populations in the same continent. Thus, the number of SNPs in isolated populations that need to be genotyped may be only slightly less than in non-isolates. Conclusion: The "isolated population" label by itself does not guarantee a greater genotyping efficiency in association studies, and properties other than increased linkage disequilibrium may make these populations interesting in genetic epidemiology.

Transcriptional regulation of MDR1, a gene involved in antifungal drug resistance in Candida albicans

ABSTRACT Upregulation of the Major Facilitator transporter gene MDR1 (Multi_drug Resistance 1) is one of the mechanisms observed in Candida albicans clinical isolates developing resistance to azole antifungal agents. To better understand this phenomenon, the cis-acting regulatory elements present in a modulatable reporter system under the control of the MDR1 promoter were characterized. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H2O2, whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements, that are necessary and sufficient to convey the same transcriptional responses to a heterologous promoter (CDR2), were identified within the MDR1promoter. The first element, called BRE (for Benomyl Response Element, -296 to -260 with respect to the ATG start codon), is required for benomyl-dependent MDR1 upregulation and for constitutive high expression of MDR1 in FR2. The second element, termed HRE (for H2O2 Response Element, -561 to -520), is required for H2O2-dependent MDR1 upregulation, but is dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the blip transcription factor Cap1p lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H2O2 and diminished significantly the transient response to benomyl. Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a transacting and positive regulatory role in benomyl- and H2O2-dependent transcription of MDR1. However, it is not the only transcription factor involved in the response of MDR1 to benomyl. A minimal BRE element (-290 to -273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also delimited. Genome-wide transcript profiling analyses undertaken with a matched pair of clinical isolates, one of which being azole-resistant and upregulating MDR1, and with an azole-susceptible strain exposed to benomyl, revealed that genes specifically upregulated by benomyl harbour in their promoters Cap1p binding site(s). This strengthened the idea that Cap1p plays a role in benomyl-dependent upregulation of MDR1. BRE-like sequences were also identified in several genes co-regulated with MDR1 in both conditions, which was consistent with the involvement of the BRE in both processes. A set of 147 mutants lacking a single transcription factor gene was next screened for loss of MDR1response to benomyl. Unfortunately, none of the tested mutants showed a loss of benomyl-dependent MDR1 upregulation. Nevertheless, a significant diminution of the response was observed in the mutants in which the MADS-box transcription factor Mcm1p and the C2H2 zinc finger transcription factor orf19.13374p were inactivated, suggesting that Mcm1p and orf19.13374p are involved in MDR1response to benomyl. Interestingly, the BRE contains a perfect match to the binding consensus of Mcm1p, raising the possibility that MDR1may be a direct target of this transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools we have developed during characterization of the cis-acting elements of the MDR1promoter should now serve to elucidate the nature of the components that modulate its activity. RESUME La surexpression du gène MDR1 (pour Résistance Multidrogue 1), qui code pour un transporteur de la famille des Major Facilitators, est l'un des mécanismes observés dans les isolats cliniques de la levure Candida albicans développant une résistance aux agents antifongiques appelés azoles. Pour mieux comprendre ce phénomène, les éléments de régulation agissant en cis dans un système rapporteur modulable sous le contrôle du promoteur MDR1 ont été caractérisés. Dans une souche sensible aux azoles, la transcription de ce rapporteur est transitoirement surélevée en réponse soit au bénomyl soit à l'agent oxydant H2O2, alors que son expression est constitutivement élevée dans une souche résistante aux azoles (souche FR2). Deux éléments de régulation agissant en cis, nécessaires et suffisants pour transmettre les mêmes réponses transcriptionnelles à un promoteur hétérologue (CDR2), ont été identifiés dans le promoteur MDR1. Le premier élément, appelé BRE (pour Elément de Réponse au Bénomyl, de -296 à -260 par rapport au codon d'initiation ATG) est requis pour la surexpression de MDR1dépendante du bénomyl et pour l'expression constitutive de MDR1 dans FR2. Le deuxième élément, appelé HRE (pour Elément de Réponse à l'H2O2, de -561 à -520), est requis pour la surexpression de MDR1 dépendante de l'H2O2, mais n'est pas impliqué dans l'expression constitutive du gène MDR1. Deux sites de fixation potentiels (TTAG/CTAA) pour le facteur de transcription Cap1p ont été identifiés dans l'élément HRE. De plus, l'inactivation de CAP1 abolit la réponse transitoire à l'H2O2 et diminua significativement la réponse transitoire au bénomyl. Cap1p, qui est impliqué dans les réponses de la cellule au stress oxydatif, doit donc jouer un rôle positif en trans dans la surexpression de MDR1 dépendante du bénomyl et de l'H2O2. Cependant, ce n'est pas le seul facteur de transcription impliqué dans la réponse au bénomyl. Un élément BRE d'une longueur minimale (de -290 à -273) a également été défini et est suffisant pour détecter une interaction spécifique in vitro avec des protéines provenant d'extraits bruts de C. albicans. L'analyse du profil de transcription d'une paire d'isolats cliniques comprenant une souche résistante aux azoles surexprimant MDR1, et d'une souche sensible aux azoles exposée au bénomyl, a révélé que les gènes spécifiquement surexprimés par le bénomyl contiennent dans leurs promoteurs un ou plusieurs sites de fixation pour Cap1p. Ceci renforce l'idée que Cap1p joue un rôle dans la surexpression de MDR1dépendante du bénomyl. Une ou deux séquences ressemblant à l'élément BRE ont également été identifiées dans la plupart des gènes corégulés avec MDR1 dans ces deux conditions, ce qui était attendu compte-tenu du rôle joué par cet élément dans les deux processus. Une collection de 147 mutants dans lesquels un seul facteur de transcription est inactivé a été testée pour la perte de réponse au bénomyl de MDR1. Malheureusement, la surexpression de MDR1 dépendante du bénomyl n'a été perdue dans aucun des mutants testés. Néanmoins, une diminution significative de la réponse a été observée chez des mutants dans lesquels le facteur de transcription à MADS-box Mcm1p et le facteur de transcription à doigts de zinc de type C2H2 orf19.13374p ont été inactivés, suggérant que Mcm1p et orf19.13374p sont impliqués dans la réponse de MDR1au bénomyl. Il est intéressant de noter que la BRE contient une séquence qui s'aligne parfaitement avec la séquence consensus du site de fixation de Mcm1p, ce qui soulève la possibilité que MDR1 pourrait être une cible directe de cet activateur transcriptionnel. En conclusion, alors que l'identité des facteurs agissant en trans en se fixant à la BRE et à la HRE reste à être confirmée, les outils que nous avons développés au cours de la caractérisation des éléments agissant en cis sur le promoteur MDR1 peut maintenant servir à élucider la nature des composants modulant son activité.

In vitro effect of malachite green on Candida albicans involves multiple pathways and transcriptional regulators UPC2 and STP2.

In this study, we show that a chemical dye, malachite green (MG), which is commonly used in the fish industry as an antifungal, antiparasitic, and antibacterial agent, could effectively kill Candida albicans and non-C. albicans species. We have demonstrated that Candida cells are susceptible to MG at a very low concentration (MIC that reduces growth by 50% [MIC(50)], 100 ng ml(-1)) and that the effect of MG is independent of known antifungal targets, such as ergosterol metabolism and major drug efflux pump proteins. Transcriptional profiling in response to MG treatment of C. albicans cells revealed that of a total of 207 responsive genes, 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, heat shock, amino acid metabolism, etc., were upregulated, while 37 genes involved in iron acquisition, filamentous growth, mitochondrial respiration, etc., were downregulated. We confirmed experimentally that Candida cells exposed to MG resort to a fermentative mode of metabolism, perhaps due to defective respiration. In addition, we showed that MG triggers depletion of intracellular iron pools and enhances reactive oxygen species (ROS) levels. These effects could be reversed by the addition of iron or antioxidants, respectively. We provided evidence that the antifungal effect of MG is exerted through the transcription regulators UPC2 (regulating ergosterol biosynthesis and azole resistance) and STP2 (regulating amino acid permease genes). Taken together, our transcriptome, genetic, and biochemical results allowed us to decipher the multiple mechanisms by which MG exerts its anti-Candida effects, leading to a metabolic shift toward fermentation, increased generation of ROS, labile iron deprivation, and cell necrosis.

Increased endometrial placenta growth factor (PLGF) gene expression in women with successful implantation.

Objective: To analyze the vascularization of the endometrium via hysteroscopy and to assess its correlation with angiogenic factor gene expression and embryo implantation rate.Design: Cross-sectional study.Setting: Public university hospital.Patient(s): Patients undergoing hysteroscopy for supposed infertility.Intervention(s): Endometrial quality assessment according to Sakumoto-Masamoto, performed in the early secretory phase of the cycle. Collection of an endometrial tissue biopsy.Main Outcome Measure(s): RNA extraction, reverse transcription, and determination of gene expression of angiogenesis- and implantation-relevant factors using quantitative polymerase chain reaction. Retrieval of pregnancy information from the medical records.Result(s): Good quantity/quality RNA with infertility history was obtained from 63 participating women. Those with a "good" endometrium and subsequent pregnancy showed increased gene expression for placenta growth factor when compared with patients with a "bad" endometrium and who did not succeed with pregnancy to date. Nonpregnant women with a "good" endometrium presented an intermediate result. No significant differences were observed for several other genes tested, but trends in the same direction were observed.Conclusion(s): This study demonstrates for the first time that endometrial PLGF expression corresponds to the hysteroscopic appearance of the endometrium, and therefore has potential as a clinically relevant prognosticator for infertility treatment success. (Fertil Steril (R) 2011;96:663-8. (C)2011 by American Society for Reproductive Medicine.)

Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0.

AIMS: To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. METHODS AND RESULTS: Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. CONCLUSIONS: The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.

Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides

OBJECTIVE: A distinct subset of proinflammatory CD4+ T cells that produce interleukin-17 was recently identified. These cells are implicated in different autoimmune disease models, such as experimental autoimmune encephalomyelitis and collagen-induced arthritis, but their involvement in human autoimmune disease has not yet been clearly established. The purpose of this study was to assess the frequency and functional properties of Th17 cells in healthy donors and in patients with different autoimmune diseases. METHODS: Peripheral blood was obtained from 10 psoriatic arthritis (PsA), 10 ankylosing spondylitis (AS), 10 rheumatoid arthritis (RA), and 5 vitiligo patients, as well as from 25 healthy donors. Synovial tissue samples from a separate group of patients were also evaluated (obtained as paraffin-embedded sections). Peripheral blood cells were analyzed by multiparameter flow cytometry and immunohistochemistry. Cytokine production was examined by enzyme-linked immunosorbent assay and intracellular cytokine staining using specific monoclonal antibodies. Synovial tissue was examined for infiltrating T cells by immunohistochemical analysis. RESULTS: We found increased numbers of circulating Th17 cells in the peripheral blood of patients with seronegative spondylarthritides (PsA and AS), but not in patients with RA or vitiligo. In addition, Th17 cells from the spondylarthritis patients showed advanced differentiation and were polyfunctional in terms of T cell receptor-driven cytokine production. CONCLUSION: These observations suggest a role of Th17 cells in the pathogenesis of certain human autoimmune disorders, in particular the seronegative spondylarthritides.

Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods.

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Delta/cdr1Delta, cdr2Delta/cdr2Delta, flu1Delta/flu1Delta, and mdr1Delta/mdr1Delta) and calcineurin subunit A (cnaDelta/cnaDelta) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% +/- 5.0%/96.5% +/- 2.4% (HPLC) and 94.9% +/- 6.1%/94.7% +/- 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% +/- 1.5%/5.4% +/- 0.9% and 6.5% +/- 2.5%/4.0% +/- 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg x h/liter (range, 12.9 to 71.1) on the first and 27.4 mg x h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg x h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg x h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.

Increased plasma levels of N-terminal brain natriuretic peptide (NT-proBNP) in type 2 diabetic patients with vascular complications

Résumé : Les concentrations plasmatiques du peptide natriurétique de type B sont augmentées chez les diabétiques de type 2 atteints de complications vasculaires. But : Les concentrations plasmatiques du peptide natriurétique de type B (NT-proBNP) sont augmentées chez les diabétiques de type 2 atteints de complications vasculaires. Les concentrations plasmatiques du peptide natriurétique de type B (BNP), ou de sa pro-hormone (NT-proBNP), sont reconnues depuis peu comme marqueur de choix de la dysfonction cardiaque. Les diabétiques de type 2 sont à haut risque de développer des complications cardiovasculaires. L'objectif de cette étude a été de déterminer si les concentrations plasmatiques de NT-proBNP étaient comparables chez des diabétiques de type 2 avec ou sans complications vasculaires. Méthodes : Nous avons mesuré le NT-proBNP plasmatique chez 54 diabétiques de type 2, 27 sans complications micro- ou macrovasculaires et 27 présentant des complications soit micro- soit macrovasculaires, soit les deux. Le même dosage a été effectué chez 38 témoins sains, appariés pour l'âge et le sexe avec les diabétiques. Résultat : Le NT-proBNP plasmatique était plus élevé chez les diabétiques avec complications (médiane 121 pg/ml, intervalle interquartile 50-240 pg/ml) que chez ceux sans complications (37 pg/ml, 21-54 pg/ml, P < 0,01). Comparés au groupe témoin (55 pg/ml, 40-79 pg/ml), seuls les diabétiques avec complications vasculaires avaient un NT-proBNP plasmatique significativement augmenté (P < 0,001). Chez les diabétiques la maladie coronarienne et la néphropathie (définie selon l'excrétion urinaire d'albumine) étaient chacune associée de façon indépendante avec une augmentation des concentrations plasmatiques de NT-proBNP. Conclusion : Chez les diabétiques de type 2 souffrant de complications micro- ou macrovasculaires, les concentrations plasmatiques de NT-proBNP sont augmentées par rapport à celles des malades indemnes de complications vasculaires. L'augmentation de sécrétion de ce peptide est associée de façon indépendante avec la maladie coronarienne et la néphropathie. La mesure du NT-proBNP plasmatique pourrait donc être utile pour dépister la présence de complications micro- ou macrovasculaires.

Epidermal growth factor (EGF) stimulation of cultured brain cells. II. Increased production of extracellular soluble proteins.

The production of extracellular soluble proteins was studied in serum-free aggregating cell cultures of fetal rat telencephalon labeled on culture day 7 with a mixture of radioactive amino acid precursors. Cultures treated continuously with epidermal growth factor (EGF; 20 ng/ml) showed a generally increased protein secretion and a particularly enhanced production of a few distinct extracellular proteins. The time lag of this response after an initial dose of EGF (25 ng/ml) on day 7 was 48 h. The total macromolecular radioactivity that accumulated within 96 h of labeling in the media of EGF-treated cultures was 175% of untreated controls, whereas no difference was found in the proportions of intracellular amino acid incorporation. Cultures which received a single dose of EGF (25 ng/ml) on day 1 showed still a greatly increased protein secretion on day 7. Prevention of extracellular protein accumulation by reducing the initial cell number and increasing the rate of media changes did not affect the EGF-induced stimulation of the two glial enzymes, glutamine synthetase and 2',3'-cyclic nucleotide 3'-phosphohydrolase. The results suggest that both the increased production of extracellular proteins and the enhanced development of glial enzymatic activities reflect the stimulated phenotypic expression of EGF-sensitive brain cells.

Pivotal role for a tail subunit of the RNA polymerase II mediator complex CgMed2 in azole tolerance and adherence in Candida glabrata.

Antifungal therapy failure can be associated with increased resistance to the employed antifungal agents. Candida glabrata, the second most common cause of invasive candidiasis, is intrinsically less susceptible to the azole class of antifungals and accounts for 15% of all Candida bloodstream infections. Here, we show that C. glabrata MED2 (CgMED2), which codes for a tail subunit of the RNA polymerase II Mediator complex, is required for resistance to azole antifungal drugs in C. glabrata. An inability to transcriptionally activate genes encoding a zinc finger transcriptional factor, CgPdr1, and multidrug efflux pump, CgCdr1, primarily contributes to the elevated susceptibility of the Cgmed2Δ mutant toward azole antifungals. We also report for the first time that the Cgmed2Δ mutant exhibits sensitivity to caspofungin, a constitutively activated protein kinase C-mediated cell wall integrity pathway, and elevated adherence to epithelial cells. The increased adherence of the Cgmed2Δ mutant was attributed to the elevated expression of the EPA1 and EPA7 genes. Further, our data demonstrate that CgMED2 is required for intracellular proliferation in human macrophages and modulates survival in a murine model of disseminated candidiasis. Lastly, we show an essential requirement for CgMed2, along with the Mediator middle subunit CgNut1 and the Mediator cyclin-dependent kinase/cyclin subunit CgSrb8, for the high-level fluconazole resistance conferred by the hyperactive allele of CgPdr1. Together, our findings underscore a pivotal role for CgMed2 in basal tolerance and acquired resistance to azole antifungals.