Effects of neuropeptide Y on the blood pressure response to various vasoconstrictor agents.

Neuropeptide Y (NPY) is known to potentiate the pressor effect of norepinephrine. In the present work, we evaluated in unanesthetized normotensive rats the effect of NPY on blood pressure responsiveness not only to norepinephrine, but also to tyramine, a sympathomimetic agent acting indirectly to B-HT933, a selective alpha-2 adrenoceptor stimulant, to angiotensin II and vasopressin. Dose-response curves to the various pressor agents were established starting at the 45th min of an i.v. infusion with either NPY (0.025 and 0.1 microgram/min) or its vehicle. The two doses of NPY increased blood pressure by an average of approximately 6 mm Hg, which was not significantly different from the vehicle-induced blood pressure changes. NPY significantly enhanced the pressor effect of norepinephrine, tyramine and angiotensin II, but not that of B-HT933 and vasopressin. We also tested whether NPY inhibits the enzyme activity of Na, K-adenosine triphosphatase using a purified toad kidney preparation. Concentrations of NPY from 10(-14) M up to 10(-6) M had no effect on the enzyme activity. It appears therefore that the blood pressure potentiating effect of NPY is not restricted to alpha adrenoceptor stimulation with norepinephrine, but involves also the vasoconstrictor hormone angiotensin II. This NPY-induced potentiation does not seem to depend upon stimulation of alpha-2 adrenoceptors or inhibition of Na,K-adenosine triphosphatase.

The alarmin HMGB1 enhances CXCR4-induced activities by binding CXCL12

A variety of chemokines and inflammatory molecules are concomitantly produced at target sites of leukocyte trafficking and homing, accounting for the complex cellular responses occurring in homeostasis and inflammation. The chemokine CXCL12 plays an essential and unique role in homeostatic regulation of leukocyte traffic and tissue regeneration. The chromatin protein HMGB1 is released by dying and distressed cells, and acts as a Damage Associated Molecular Pattern or alarmin, promoting cell migration towards the site of tissue damage. We show here that HMGB1 synergises with CXCL12 by forming a heterocomplex that we characterized by NMR chemical shift mapping. The heterocomplex enhances CXCR4-induced responses on cells of the immune system, acting exclusively through the CXCL12 receptor CXCR4, and not through the HMGB1 receptors RAGE, TLR2 and TLR4. FRET analysis show that CXCL12 and CXCL12+HMGB1 promote a different conformational change in the homodimer CXCR4. The enhancement induced by HMGB1 on CXCL12-induced migration is selective, since little changes in migration of neutrophils and PreB 300.19-CCR2+ or -CCR7+ are observed towards CXCL8 and CCR2 or CCR7 agonists. HMGB1 also promotes CXCL 12 release, which is ultimately responsible for the chemoattractant activities of HMGB1. This study highlights the role of HMGB1 in promoting CXCL12-dependent cell migration, and suggests a cooperative role of these two molecules in tissue repair as well as in pathological conditions, such as rheumatoid arthritis.

Begging food provisioning and nestling competition in great tit broods infested with ectoparasites

Ectoparasites are a ubiquitous environmental component of breeding birds, and it has repeatedly been shown that hematophagous ectoparasites such as fleas and mites reduce the quality and number of offspring of bird hosts, thereby lowering the value of a current brood. Selection acting on the hosts will favor physiological and behavioral responses that will reduce the parasites' impact. However, the results of the few bird studies that addressed the question of whether parasitism leads to a higher rate of food provisioning are equivocal, and the begging response to infestation has rarely been quantified. A change in begging activity and parental rate of food provisioning could be predicted in either direction: parents could reduce their investment in the brood in order to invest more in future broods, or they could increase their investment in order to compensate for the parasites' effect on the current brood. Since the nestlings are weakened by the ectoparasites they may beg less, but on the other hand they may beg more in order to obtain more food. In this study we show experimentally that (1) hen fleas (Ceratophyllus gallinae) reduce the body mass and size of great tit (Parus major) nestlings, (2) nestlings of parasitized broods more than double their begging rate, (3) the male parents increase the frequency of feeding trips by over 50%, (4) the females do not adjust feeding rate to the lowered nutritional state of nestlings, and (5) food competition among siblings of parasitized broods is increased. Ultimately the difference in the parental feeding response may be understood as the result of a sex-related difference in the trade-off of investing in current versus future broods.

Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells.

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

Role of notch signalling in mammary gland

ÁBSTRACT : Mammary gland is composed of two main epithelial cell types, myoepithelial and luminal. The mechanisms involved in determination and maintenance of them remain poorly understood. Notch signaling is known to regulate cell fate determination in other tissues like skin and nervous system. It was also shown that it can act as tumor suppressor or oncogene depending on the tissue type. The mouse models overexpressing active Notch receptors indicated that Notch signaling is oncogenic in the mammary gland. This observation was followed by some descriptive and functional studies in human breast cancer and it was reported that Notch signaling activity or expression of its components are increased in some of the breast tumor samples compared to normal tissue. However, the physiological role of the Notch signaling and its downstream mechanisms in mammary gland is poorly defined. p63, a member of p53 family, has been implicated in the cell fate determination of keratinocytes. Knockout mouse models revealed that p63 is required for the formation of the mammary anlagen in embryo and its ΔN isoform is expressed exclusively in the myoepithelial layer of the adult breast. In order to understand its function in normal breast epithelial cells, I activated Notch signaling by expression of Notch1 intracellular domain (NICD) in normal primary human breast epithelial cells (HBECs). In this context, NICD reduced growth of HBECs and led to downmodulation of extracellular matrix-receptor interaction network (ECM) components as well as ΔNp63. Expression of ΔNp63 together with NICD partially rescued Notch induced growth reduction, which was correlated with an increase in ECM components. Moreover, silencing ΔNp63 in myoepithelial HBECs reduced growth similar to Notch activation and it led to downregulation of myoepithelial and upregulation of luminal markers. Complementing this observation, forced expression of ONp63 in luminal HBECs induced myoepithelial phenotype and decreased luminal markers. In vivo, by the analysis of a Notch reporter mouse strain, I showed that Notch is activated during puberty specifically at the sites of ductal morphogenesis, terminal end buds. FAGS analysis revealed that it can be detected in two different populations based on CD24 expression (low (lo) or high (high)): at lower levels in CD24lo, which includes stem/progenitor and myoepithelial cells and higher levels in CD24hi, which contains luminal cells. In parallel with in vitro results, the CD24lo mouse mammary epithelial cells displaying Notch activity have lower levels of p63 expression. Furthermore, deletion of RBPjk, the main mediator of Notch signaling, or the overexpression of ΔNp63 inhibited luminal cell lineage in vivo. Another important point revealed by Notch reporter mouse strain is the simultaneous activation of Notch with estrogen signaling during pubertal development. The expression of FOXA1, the mediator of estrogen receptor (ER) transcriptional activity, is correlated with Notch activation in vivo that it is lower in CD24lo than in CD24hi cells. Moreover, FOXA1 is regulated by NICD in vitro supporting the presence of a link between Notch and ER signaling. Taken together, I report that Notch signaling is involved in luminal cell fate determination and its effects are partially mediated through inhibition of ONp63. Besides, ΔNp63 is required for the maintenance and sufficient for the induction of myoepithelial phenotype in HBECs in vitro and is not compatible with luminal lineage in vivo. Based on these results, I propose a model for epithelial cell hierarchy in mammary gland, whereby there are two different types of luminal progenitors, early and late, displaying different levels of Notch activity. Notch signaling contributes to the determination of luminal cell lineage in these two progenitor steps: In "Early Luminal Progenitor" stage, it inhibits myoepithelial fate by decreasing p63 expression, and in "Late Luminal Progenitor" stage, Notch signaling is involved in induction of luminal lineage by acting on ER-FOXA1 axis. It has to be investigated further whether Notch signaling might behave as an oncogene or tumor suppressor depending on which cell type in the epithelial hierarchy it is modulated and which one is more likely to occur in different human breast cancer types. RÉSUMÉ : La glande mammaire est composée de deux types principaux de cellules: les cellules luminales, qui bordent le lumen et les cellules myoépithéliales, qui se trouvent entre la lame basale et les cellules luminales. Les mécanismes intervenant dans leur différenciation et leur maintenance demeurent encore mal compris. La protéine transmembranaire Notch est connue pour déterminer le destin des cellules dans plusieurs types de tissus comme la peau ou le système nerveux. Selon le type de tissu dans lequel se trouve Notch, il agira soit comme un suppresseur de tumeur soit comme un oncogène. A l'aide de modèles de souris surexprimant les récepteurs actifs de Notch, il a été démontré que la voie de signalisation de Notch est oncogénique au niveau de la glande mammaire. Des études descriptives et fonctionnelles dans le cadre du cancer du sein ont permis de mettre en évidence une augmentation de l'activité de Notch ou de l'expression de ces composants dans certains tissus cancéreux. Toutefois, le rôle physiologique de Notch et des mécanismes qu'il active restent méconnus. P63, une protéine membre de la famille p53, est impliquée dans la différenciation des kératinocytes. Le modèle issu de l'étude des souris p63 knockout a révélé que cette protéine est requise pour la formation des primordia mammaires chez l'embryon et que son isoforme ΔNp63 est exclusivement exprimée dans la couche myoépithéliale de la glande mammaire adulte. Dans le but de comprendre les fonctions physiologiques de Notch, je l'ai activé en exprimant le domaine intracellulaire de Notch 1 (NICD) dans des cellules épithéliales primaires de glande mammaire humaine (HBECs). Le NICD a alors réduit la croissance des HBECs et conduit à la régulation négative non seulement de p63 mais également des composants du réseau d'interaction des récepteurs de la matrice extracellulaire (ECM). En exprimant conjointement ΔNp63 et NICD, il est apparu que la réduction de croissance induite par Notch était partiellement compensée, et qu'il y avait également une augmentation des composants ECM. De plus, lorsque ΔNp63 a été inactivé dans les cellules HBECs myoépithéliales, une réduction de croissance cellulaire identique à celle provoquée par l'activation de Notch a pu être mise en évidence, de même qu'une régulation négative des marqueurs myoépithéliaux ainsi qu'une augmentation des marqueurs luminaux. Afin de compléter ces informations, l'expression de ΔNp63 a été forcée dans les HBECs luminales, ce qui a induit un phénotype myoépithélial et une diminution des marqueurs lumineux. In vivo, par l'analyse de souris ayant un gène rapporteur de l'activité de Notch, j'ai démontré que Notch est activé pendant la puberté au niveau des sites de la morphogenèse canalaire, à savoir les bourgeons terminaux. Les analyses par FACS (Fluorescence-activated cell sorting) basées sur l'expression de l'antigène CD24 ont révélé qu'il peut tre détecté dans deux populations différentes : une population qui l'exprime faiblement, qui regroupe les cellules souches/progéniteurs et les cellules myoépithéliales, et une population qui l'exprime fortement qui est composé des cellules luminales. Parallèlement aux résultats in vitro, j'ai mis en évidence un faible niveau d'expression de p63 dans les cellules épithéliales de la glande mammaire de souris, exprimant faiblement l'antigène CD24 et présentant une activité de Notch. De plus, la délétion de RBPjr~, médiateur principal de la signalisation de Notch, ainsi que la surexpression de ΔNp63 in vivo ont inhibé la lignée des cellules luminales. Un autre point important révélé par les souris rapporteur de l'activité de Notch a été l'activation simultanée de Notch et de la signalisation de l'oestrogène pendant le développement pubertaire. L'expression de FOXA1, médiateur de l'activité transcriptionnelle des récepteurs aux oestrogènes (ER), est en corrélation avec l'activation de Notch in vivo, plus basse dans les cellules avec une faible expression de l'antigène CD24 que dans celles avec une forte expression. De plus, FOXA1 est régulé par NICD in vitro confirmant la présence d'un lien entre Notch et la signalisation des ER. En résumé, la signalisation de Notch est impliquée dans la détermination du destin cellulaire des cellules luminales et ses effets sont partiellement modifiés par l'inhibition de ΔNp63. ΔNp63 est requis pour la maintenance et est suffisant pour l'induction du phénotype myoépithéliale dans les HBECs in vitro et ne peut donc pas se trouver dans les cellules luminales in vivo. Basé sur ces résultats, je propose un modèle de hiérarchisation des cellules épithéliales de la glande mammaire, dans lequel sont présents deux types de progéniteurs des cellules luminales exprimant des niveaux différents d'activité de Notch, les progéniteurs lumineux précoces et tardifs. La signalisation de Notch contribue à la différenciation de la lignée cellulaire luminale au niveau de ces deux progéniteurs : dans la forme précoce, il inhibe la différenciation des cellules myoépithéliales en réduisant l'expression de p63 et dans la forme tardive, Notch est impliqué dans l'induction de la lignée luminale en agissant sur l'axe ER-FOXA1. Il serait nécessaire d'investiguer plus loin si le fait que Notch agisse comme oncogène ou suppresseur de tumeur dépend du stade de différenciation de la cellule dans laquelle il est modulé et laquelle de ces deux fonctions il est le plus probable de rencontrer dans les différents types de cancer du sein.

Chirurgie du diabète: provocation ou réalité [Surgery for diabetes: challenge or reality?]

Bariatric surgery techniques can usually increase either sensitivity or insulin resistance, acting on three different levels: decrease of food intake, malabsorption and modifications of entero-insulaire axis activity. This latter is taken into account in order to develop a new protocol to obtain diabetes remission without important weight loss and nutritional deficiencies. Preliminary results are interesting but they come from very short time studies with few patients. Moreover, complications rate is at present very high. Knowing better gastrointestinal mechanisms of diabetes control and especially incretins role is absolutely necessary before identifying surgery as a true metabolic treatment.

Homeodomain binding motifs modulate the probability of odorant receptor gene choice in transgenic mice.

Odorant receptor (OR) genes constitute with 1200 members the largest gene family in the mouse genome. A mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and from one allele. The cell bodies of OSNs that express a given OR gene display a mosaic pattern within a particular region of the main olfactory epithelium. The mechanisms and cis-acting DNA elements that regulate the expression of one OR gene per OSN - OR gene choice - remain poorly understood. Here, we describe a reporter assay to identify minimal promoters for OR genes in transgenic mice, which are produced by the conventional method of pronuclear injection of DNA. The promoter transgenes are devoid of an OR coding sequence, and instead drive expression of the axonal marker tau-β-galactosidase. For four mouse OR genes (M71, M72, MOR23, and P3) and one human OR gene (hM72), a mosaic, OSN-specific pattern of reporter expression can be obtained in transgenic mice with contiguous DNA segments of only ~300 bp that are centered around the transcription start site (TSS). The ~150bp region upstream of the TSS contains three conserved sequence motifs, including homeodomain (HD) binding sites. Such HD binding sites are also present in the H and P elements, DNA sequences that are known to strongly influence OR gene expression. When a 19mer encompassing a HD binding site from the P element is multimerized nine times and added upstream of a MOR23 minigene that contains the MOR23 coding region, we observe a dramatic increase in the number of transgene-expressing founders and lines and in the number of labeled OSNs. By contrast, a nine times multimerized 19mer with a mutant HD binding site does not have these effects. We hypothesize that HD binding sites in the H and P elements and in OR promoters modulate the probability of OR gene choice.

La stratégie thérapeutique anti-VEGF améliore le pronostic visuel de la dégénérescence maculaire liée à l'âge exsudative [Intravitreal anti-VEGF injections improve the visual prognosis of wet age related macular degeneration].

Age related macular degeneration (AMD) is an ocular disease with high prevalence among elderly persons. Two different forms exist: dry AMD, usually slowly progressive, and neovascular AMD (wet form) more aggressive. Photodynamic therapy is used to treat the wet form and anti VEGF treatments recently became available and offer a real change in the prognostic of wet AMD. Two products are registered and used in Switzerland (Macugen and Lucentis), a third "off labels product", Avastin is also currently used in clinical practice. Nevertheless, both the duration of treatment and the number of injection requested to stabilise the disease were not defined in the studies. Ongoing studies are mainly evaluating combined treatments and long acting form of the drug.

Fatal tolperisone poisoning: Autopsy and toxicology findings in three suicide cases.

Tolperisone (Mydocalm(®)) is a centrally acting muscle relaxant with few sedative side effects that is used for the treatment of chronic pain conditions. We describe three cases of suicidal tolperisone poisoning in three healthy young subjects in the years 2006, 2008 and 2009. In all cases, macroscopic and microscopic autopsy findings did not reveal the cause of death. Systematic toxicological analysis (STA) including immunological tests, screening for volatile substances and blood, urine and gastric content screening by GC-MS and HPLC-DAD demonstrated the presence of tolperisone in all cases. In addition to tolperisone, only the analgesics paracetamol (acetaminophen), ibuprofen and naproxen could be detected. The blood ethanol concentrations were all lower than 0.10g/kg. Tolperisone was extracted by liquid-liquid extraction using n-chlorobutane as the extraction solvent. The quantification was performed by GC-NPD analysis of blood, urine and gastric content. Tolperisone concentrations of 7.0mg/l, 14mg/l and 19mg/l were found in the blood of the deceased. In the absence of other autopsy findings, the deaths in these three cases were finally explained as a result of lethal tolperisone ingestion. To the best of our knowledge, these three cases are the first reported cases of suicidal tolperisone poisonings.

Adaptive diversity in heterogeneous environments for populations regulated by a mixture of soft and hard selection

The stable co-existence of two haploid genotypes or two species is studied in a spatially heterogeneous environment submitted to a mixture of soft selection (within-patch regulation) and hard selection (outside-patch regulation) and where two kinds of resource are available. This is analysed both at an ecological time-scale (short term) and at an evolutionary time-scale (long term). At an ecological scale, we show that co-existence is very unlikely if the two competitors are symmetrical specialists exploiting different resources. In this case, the most favourable conditions are met when the two resources are equally available, a situation that should favour generalists at an evolutionary scale. Alternatively, low within-patch density dependence (soft selection) enhances the co-existence between two slightly different specialists of the most available resource. This results from the opposing forces that are acting in hard and soft regulation modes. In the case of unbalanced accessibility to the two resources, hard selection favours the most specialized genotype, whereas soft selection strongly favours the less specialized one. Our results suggest that competition for different resources may be difficult to demonstrate in the wild even when it is a key factor in the maintenance of adaptive diversity. At an evolutionary scale, a monomorphic invasive evolutionarily stable strategy (ESS) always exists. When a linear trade-off exists between survival in one habitat versus that in another, this ESS lies between an absolute adjustment of survival to niche size (for mainly soft-regulated populations) and absolute survival (specialization) in a single niche (for mainly hard-regulated populations). This suggests that environments in agreement with the assumptions of such models should lead to an absence of adaptive variation in the long term.

Rhumatologie. Denosumab: le joker du rhumatologue [Denosumab, the rheumatologist's joker?]

The number of molecules acting on bone turn over rapidly increasing. The idea to use them on bones erosions is not new, however this year a new molecule confirms the suitability of such an approach with the demonstration of efficency in both postmenopausal osteoporosis as well as in the prevention of bone erosions in rheumatoid arthritis. Denosumab, a human monoclonal antibody against RANKL (Receptor Activator of Nuclear factor-KB ligand), decreases the fracture risk in postmenopausal osteoporosis and prevents new bone erosions in rheumatoid arthritis. Of simple use, it appears to act rapidly, to be efficient with a sustain benefit. The tolerance seems excellent, and now we'll have just to wait for its licensing.

Differential involvement of MEK kinase 1 (MEKK1) in the induction of apoptosis in response to microtubule-targeted drugs versus DNA damaging agents.

MEK kinase 1 (MEKK1) is a 196-kDa enzyme that is involved in the regulation of the c-Jun N-terminal kinase (JNK) pathway and apoptosis. In cells exposed to genotoxic agents including etoposide and cytosine arabinoside, MEKK1 is cleaved at Asp874 by caspases. The cleaved kinase domain of MEKK1, itself, stimulates caspase activity leading to apoptosis. Kinase-inactive MEKK1 expressed in HEK293 cells effectively blocks genotoxin-induced apoptosis. Treatment of cells with taxol, a microtubule stabilizing agent, did not induce MEKK1 cleavage in cells, and kinase-inactive MEKK1 expression failed to block taxol-induced apoptosis. MEKK1 became activated in HEK293 cells exposed to taxol, but in contrast to etoposide-treatment, taxol failed to increase JNK activity. Taxol treatment of cells, therefore, dissociates MEKK1 activation from the regulation of the JNK pathway. Overexpression of anti-apoptotic Bcl2 blocked MEKK1 and taxol-induced apoptosis but did not block the caspase-dependent cleavage of MEKK1 in response to etoposide. This indicates Bcl2 inhibition of apoptosis is, therefore, downstream of caspase-dependent MEKK1 cleavage. The results define the involvement of MEKK1 in the induction of apoptosis by genotoxins but not microtubule altering drugs.

The role of oxidative stress during inflammatory processes.

Abstract The production of various reactive oxidant species in excess of endogenous antioxidant defense mechanisms promotes the development of a state of oxidative stress, with significant biological consequences. In recent years, evidence has emerged that oxidative stress plays a crucial role in the development and perpetuation of inflammation, and thus contributes to the pathophysiology of a number of debilitating illnesses, such as cardiovascular diseases, diabetes, cancer, or neurodegenerative processes. Oxidants affect all stages of the inflammatory response, including the release by damaged tissues of molecules acting as endogenous danger signals, their sensing by innate immune receptors from the Toll-like (TLRs) and the NOD-like (NLRs) families, and the activation of signaling pathways initiating the adaptive cellular response to such signals. In this article, after summarizing the basic aspects of redox biology and inflammation, we review in detail the current knowledge on the fundamental connections between oxidative stress and inflammatory processes, with a special emphasis on the danger molecule high-mobility group box-1, the TLRs, the NLRP-3 receptor, and the inflammasome, as well as the transcription factor nuclear factor-κB.

IL28B single nucleotide polymorphisms in the treatment of hepatitis C.

Recent genome-wide association studies (GWAS) have identified genetic variations near the IL28B gene which are strongly associated with spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infection. Protective IL28B variations are strongly associated with on-treatment viral kinetics and approximately 2-fold increased sustained virologic response (SVR) rates in HCV genotype 1 and 4 patients. In HCV genotype 1 patients, IL28B variations were shown to be the strongest pre-treatment predictor of virologic response. In the treatment of HCV genotype 2 and 3 infected patients, IL28B variations play only a minor role. Preliminary data indicate that IL28B variations are also associated with treatment outcome of regimens, including directly acting antiviral (DAA) agents, though their impact seems to be attenuated compared to standard treatment. Here, we review these important findings and discuss possible implications for clinical decision making in the treatment of HCV infection.

Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections.

Matrix attachment regions are DNA sequences found throughout eukaryotic genomes that are believed to define boundaries interfacing heterochromatin and euchromatin domains, thereby acting as epigenetic regulators. When included in expression vectors, MARs can improve and sustain transgene expression, and a search for more potent novel elements is therefore actively pursued to further improve recombinant protein production. Here we describe the isolation of new MARs from the mouse genome using a modified in silico analysis. One of these MARs was found to be a powerful activator of transgene expression in stable transfections. Interestingly, this MAR also increased GFP and/or immunoglobulin expression from some but not all expression vectors in transient transfections. This effect was attributed to the presence or absence of elements on the vector backbone, providing an explanation for earlier discrepancies as to the ability of this class of elements to affect transgene expression under such conditions.