Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

Bacterial flagellin elicits widespread innate immune defense mechanisms, apoptotic signaling, and a sepsis-like systemic inflammatory response in mice.

Introduction: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. Methods: Male Balb/C mice (n = 80) were injected intravenously with 1-5 mu g recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NF kappa B) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. Results: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NF kappa B and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNF alpha, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1 beta and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. Conclusions: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organisms

Immune priming and pathogen resistance in ant queens.

Growing empirical evidence indicates that invertebrates become more resistant to a pathogen following initial exposure to a nonlethal dose; yet the generality, mechanisms, and adaptive value of such immune priming are still under debate. Because life-history theory predicts that immune priming and large investment in immunity should be more frequent in long-lived species, we here tested for immune priming and pathogen resistance in ant queens, which have extraordinarily long life span. We exposed virgin and mated queens of Lasius niger and Formica selysi to a low dose of the entomopathogenic fungus Beauveria bassiana, before challenging them with a high dose of the same pathogen. We found evidence for immune priming in naturally mated queens of L. niger. In contrast, we found no sign of priming in virgin queens of L. niger, nor in virgin or experimentally mated queens of F. selysi, which indicates that immune priming in ant queens varies according to mating status and mating conditions or species. In both ant species, mated queens showed higher pathogen resistance than virgin queens, which suggests that mating triggers an up-regulation of the immune system. Overall, mated ant queens combine high reproductive output, very long life span, and elevated investment in immune defense. Hence, ant queens are able to invest heavily in both reproduction and maintenance, which can be explained by the fact that mature queens will be protected and nourished by their worker offspring.

Reactive electrophile species activate defense gene expression in Arabidopsis.

Compounds containing alpha,beta-unsaturated carbonyl groups are increasingly implicated as potent regulators of gene expression; some are powerful cytotoxins known to accumulate at the site of lesion formation in host-pathogen interactions. We used a robust measurement of photosynthetic efficiency to quantify the toxicity of a variety of lipid derivatives in Arabidopsis leaves. Small alpha,beta-unsaturated carbonyl compounds (e.g. acrolein and methyl vinyl ketone) were highly active and proved to be potent stimulators of expression of the pathogenesis-related gene HEL (PR4). These small volatile electrophiles were far more active than larger alkenal homologs like 2(E)-hexenal, and activated HEL expression in a manner independent of salicylate, ethylene, and jasmonate production/perception. Electrophile treatment massively increased the levels of unesterified cyclopentenone jasmonates, which themselves are electrophiles. Patterns of gene expression in response to electrophile treatment and in response to avirulent bacteria were compared, which revealed strikingly similar transcript profiles. The results broaden the range of known biologic effects of reactive electrophile species to include the activation of a pathogenesis-related gene (HEL) and genes involved in metabolism. Electrophiles can act as mediators of both genetic and biochemical effects on core defense signal transduction.

Role of secretory immunoglobulin A and secretory component in the protection of mucosal surfaces.

The contribution of secretory immunoglobulin A (SIgA) antibodies in the defense of mucosal epithelia plays an important role in preventing pathogen adhesion to host cells, therefore blocking dissemination and further infection. This mechanism, referred to as immune exclusion, represents the dominant mode of action of the antibody. However, SIgA antibodies combine multiple facets, which together confer properties extending from intracellular and serosal neutralization of antigens, activation of non-inflammatory pathways and homeostatic control of the endogenous microbiota. The sum of these features suggests that future opportunities for translational application from research-based knowledge to clinics include the mucosal delivery of bioactive antibodies capable of preserving immunoreactivity in the lung, gastrointestinal tract, the genito-urinary tract for the treatment of infections. This article covers topics dealing with the structure of SIgA, the dissection of its mode of action in epithelia lining different mucosal surfaces and its potential in immunotherapy against infectious pathogens.

Immune regulation and function of the NOD-like receptors NLRC5 and NLRP3

AbstractThe vertebrate immune system is composed of the innate and the adaptive branches. Innate immune cells represent the first line of defense and detect pathogens through pattern recognition receptors (PRRs), detecting evolutionary conserved pathogen- and danger- associated molecular patterns. Engagement of these receptors initiates the inflammatory response, but also instructs antigen-specific adaptive immune cells. NOD-like receptors (NLRs) are an important group of PRRs, leading to the production of inflammatory mediators and favoring antigen presentation to Τ lymphocytes through the regulation of major histocompatibility complex (MHC) molecules.In this work we focused our attention on selected NOD-like receptors (NLRs) and their role at the interface between innate and adaptive immunity. First, we describe a new regulatory mechanism controlling IL-1 production. Our results indicate that type I interferons (IFNs) block NLRP1 and NLRP3 inflammasome activity and interfere with LPS-driven proIL-Ια and -β induction. As type I IFNs are produced upon viral infections, these anti-inflammatory effects of type I IFN could be relevant in the context of superinfections, but could also help explaining the efficacy of IFN-β in multiple sclerosis treatment.The second project addresses the role of a novel NLR family member, called NLRC5. The function of this NLR is still matter of debate, as it has been proposed as both an inhibitor and an activator of different inflammatory pathways. We found that the expression of this protein is restricted to immune cells and is positively regulated by IFNs. We generated Nlrc5-deficient mice and found that this NLR plays an essential role in Τ, NKT and, NK lymphocytes, in which it drives the expression of MHC class I molecules. Accordingly, we could show that CD8+ Τ cell-mediated killing of target lymphocytes lacking NLRC5 is strongly impaired. Moreover, NLRC5 expression was found to be low in many lymphoid- derived tumor cell lines, a mechanism that could be exploited by tumors to escape immunosurveillance.Finally, we found NLRC5 to be involved in the production of IL-10 by CD4+ Τ cells, as Nlrc5- deficient Τ lymphocytes produced less of this cytokine upon TCR triggering. In line with these observations, Mrc5-deficient CD4+ Τ cells expanded more than control cells when transferred into lymphopenic hosts and led to a more rapid appearance of colitis symptoms. Therefore, our work gives novel insights on the function of NLRC5 by using knockout mice, and strongly supports the idea that NLRs direct not only innate, but also adaptive immune responses.

No evidence for immune priming in ants exposed to a fungal pathogen.

There is accumulating evidence that invertebrates can acquire long-term protection against pathogens through immune priming. However, the range of pathogens eliciting immune priming and the specificity of the response remain unclear. Here, we tested if the exposure to a natural fungal pathogen elicited immune priming in ants. We found no evidence for immune priming in Formica selysi workers exposed to Beauveria bassiana. The initial exposure of ants to the fungus did not alter their resistance in a subsequent challenge with the same fungus. There was no sign of priming when using homologous and heterologous combinations of fungal strains for exposure and subsequent challenges at two time intervals. Hence, within the range of conditions tested, the immune response of this social insect to the fungal pathogen appears to lack memory and strain-specificity. These results show that immune priming is not ubiquitous across pathogens, hosts and conditions, possibly because of immune evasion by the pathogen or efficient social defences by the host.

The clinical and biological impact of new pathogen inactivation technologies on platelet concentrates.

Since 1990, several techniques have been developed to photochemically inactivate pathogens in platelet concentrates, potentially leading to safer transfusion therapy. The three most common methods are amotosalen/UVA (INTERCEPT Blood System), riboflavin/UVA-UVB (MIRASOL PRT), and UVC (Theraflex-UV). We review the biology of pathogen inactivation methods, present their efficacy in reducing pathogens, discuss their impact on the functional aspects of treated platelets, and review clinical studies showing the clinical efficiency of the pathogen inactivation methods and their possible toxicity.

The costs of parasitism and anti-parasitic defense in the common vole

Résumé : Les relations entre un parasite et son hôte sont avant tout marquées par le coût pour l'hôte que représente la ponction de ressources au profit du parasite et ses conséquences sur les traits d'histoires de vie de l'hôte. Pour contenir la réduction de leur valeur reproductive, les hôtes ont acquis au cours de l'évolution des mécanismes soit de lutte contre les parasites, soit de réallocations des ressources. Curieusement les effets des ectoparasites sur la biologie de mammifères ont été peu étudiés. Dans une première expérience à long terme, nous avons examiné sous un angle intégratif si les puces Nosopsyllus fasciatus affectent certains paramètres physiologiques des campagnols des champs Microtus arvalis. Nous avons également testé si les puces peuvent réduire la longévité et si oui, si ce pourrait être dû à une accélération de la sénescence. Ensuite nous avons testé si la simple activation répétée du système immunitaire comme lors d'une infestation chronique pouvait aussi réduire la longévité. Dans une dernière expérience, nous avons d'abord testé si l'infestation par des puces de jeunes campagnols au stade néonatal (21 jours) pouvait modifier leur développement et leur phénotype adulte. Puis nous avons testé si la modification du phénotype adulte est une réponse prédite et potentiellement adaptative pour minimiser les effets des puces à l'âge adulte. Nos résultats montrent que l'infestation par des puces réduit la croissance subadulte, induit une forte anémie et une immunodépression, et augmente le métabolisme de repos. De plus les puces réduisent la longévité et la taille des testicules, réduisant fortement le succès reproducteur potentiel des individus parasités. La taille finale, c'est-à-dire le développement pré-adulte, détermine en grande part la longévité. La réduction de longévité ne devrait pas être due à l'investissement au profit du système immunitaire car l'activation chronique seule du système immunitaire ne réduit pas la longévité. L'infestation néonatale retarde légèrement le développement mais surtout modifie l'hématocrite et réduit les performances locomotrices des campagnols plus de 3 mois après l'infestation. Les effets immédiats du parasitisme sur la physiologie semblent bien supérieurs comparés aux effets à long terme. Nous n'avons pas d'éléments permettant d'affirmer que le parasitisme néonatal prépare les campagnols à faire face aux puces à l'âge adulte. Au contraire, le parasitisme néonatal interagit sur le parasitisme adulte pour augmenter le métabolisme de repos. Cette thèse offre une vision intégrative des mécanismes par lesquels les puces peuvent affecter la valeur reproductive de leurs hôtes. De façon générale, ces résultats 35 montrent l'importance des puces comme force de sélection chez les campagnols. Il est indispensable de prendre en compte les ectoparasites dans l'étude de l'écologie et des dynamiques de populations chez les mammifères. Summary : The relationship between a parasite and its host is fundamentally marked by the costs for host of the withdrawals of resources by parasite and the subsequent reduction in host life-history traits. Hosts have evolved a number of strategies to reduce these costs, either by fighting against the parasite directly or by reallocating resources to reduce costs on lifetime reproductive value. The effects of ectoparasites on burrowing mammals have been scarcely studied. In a first long-term experiment, we examined how fleas Nosopsyllus fasciatus affect physiological levels of the common vole, Microtus arvalis. We also examined whether fleas reduce longevity and if so, if it is due to an early senescence pattern. Then we tested if experimental activation of the immune system by repeated injections of an antigen could result in a shorter longevity. In the last experiment, we tested if short-lasting neonatal parasitism can have long-term effects on phenotype, and if these effects could induce a predictive response to reduce damages when parasitized at the adult stage. We found that parasitism by flea reduced subadult growth, induced anaemia and immunodepression, and increased energy consumption even when resting. Moreover fleas reduce longevity and testes size associated to splenomegaly, suggesting an overall reduction in fitness but we did not find any pattern of accelerated senescence explaining the early death of parasitized voles compared to non-parasitzed. The cost of mounting an immune response throughout life does not impair longevity, suggesting that it is the cost of parasitism that limits the longevity and not the immune investment. Neonatal infestation by fleas has long-term effects on physiology and reduces motor activity more than 3 months after infestation. The modification of physiology due to long-term effects seems weak compared to the immediate effects of adult infestation. We found no evidence that neonatal parasitism prepares voles to mount a predictive adaptive response in order to reduce effects of fleas on fitness components. On the contrary, neonatal parasitism seems to worsen the effect of adult parasitism. This thesis offers an integrative view of mechanisms by which fleas affect their host at the individual level. Overall, our results demonstrate the importance of fleas as a selective force in voles. These results highlight the importance of ectoparasitism in ecology of micromarnrnals and suggest a role in the dynamic of host populations.

Epithelium-Intrinsic NAIP/NLRC4 Inflammasome Drives Infected Enterocyte Expulsion to Restrict Salmonella Replication in the Intestinal Mucosa.

The gut mucosal epithelium separates the host from the microbiota, but enteropathogens such as Salmonella Typhimurium (S.Tm) can invade and breach this barrier. Defenses against such acute insults remain incompletely understood. Using a murine model of Salmonella enterocolitis, we analyzed mechanisms limiting pathogen loads in the epithelium during early infection. Although the epithelium-invading S.Tm replicate initially, this intraepithelial replicative niche is restricted by expulsion of infected enterocytes into the lumen. This mechanism is compromised if inflammasome components (NAIP1-6, NLRC4, caspase-1/-11) are deleted, or ablated specifically in the epithelium, resulting in ∼100-fold higher intraepithelial loads and accelerated lymph node colonization. Interestingly, the cytokines downstream of inflammasome activation, interleukin (IL)-1α/β and IL-18, appear dispensable for epithelial restriction of early infection. These data establish the role of an epithelium-intrinsic inflammasome, which drives expulsion of infected cells to restrict the pathogen's intraepithelial proliferation. This may represent a general defense mechanism against mucosal infections.

A randomized controlled clinical trial evaluating the performance and safety of platelets treated with MIRASOL pathogen reduction technology.

BACKGROUND: Pathogen reduction of platelets (PRT-PLTs) using riboflavin and ultraviolet light treatment has undergone Phase 1 and 2 studies examining efficacy and safety. This randomized controlled clinical trial (RCT) assessed the efficacy and safety of PRT-PLTs using the 1-hour corrected count increment (CCI(1hour) ) as the primary outcome. STUDY DESIGN AND METHODS: A noninferiority RCT was performed where patients with chemotherapy-induced thrombocytopenia (six centers) were randomly allocated to receive PRT-PLTs (Mirasol PRT, CaridianBCT Biotechnologies) or reference platelet (PLT) products. The treatment period was 28 days followed by a 28-day follow-up (safety) period. The primary outcome was the CCI(1hour) determined using up to the first eight on-protocol PLT transfusions given during the treatment period. RESULTS: A total of 118 patients were randomly assigned (60 to PRT-PLTs; 58 to reference). Four patients per group did not require PLT transfusions leaving 110 patients in the analysis (56 PRT-PLTs; 54 reference). A total of 541 on-protocol PLT transfusions were given (303 PRT-PLTs; 238 reference). The least square mean CCI was 11,725 (standard error [SE], 1.140) for PRT-PLTs and 16,939 (SE, 1.149) for the reference group (difference, -5214; 95% confidence interval, -7542 to -2887; p<0.0001 for a test of the null hypothesis of no difference between the two groups). CONCLUSION: The study failed to show noninferiority of PRT-PLTs based on predefined CCI criteria. PLT and red blood cell utilization in the two groups was not significantly different suggesting that the slightly lower CCIs (PRT-PLTs) did not increase blood product utilization. Safety data showed similar findings in the two groups. Further studies are required to determine if the lower CCI observed with PRT-PLTs translates into an increased risk of bleeding.

Beyond splitting: observer-rated defense mechanisms in borderline personality disorder

Defense mechanism is a key concept in the psychoanalytic psychopathology of borderline personality disorder (BPD). Theoretical and empirical elaborations on this question are briefly reviewed and discussed with respect to process assessment of defense mechanisms; we put forward observer-rater methodology as an accurate means of assessing unconscious in-session processes. A sample of 25 patients presenting with BPD were interviewed, as were subjects from a matched control group without psychiatric symptoms (n = 25), using a psychodynamic interview paradigm. These interviews were transcribed and rated using the Defense Mechanisms Rating Scales. The results indicate that, compared to controls, patients with BPD used higher percentages of a action, borderline, disavowal, narcissistic, and hysteric defenses, along with lower levels of mature and obsessional defenses. Overall defensive functioning was significantly lower in the patients with BPD, compared to controls. Narcissistic defenses were related with symptom level. These results are discussed in light of previous studies on defensive functioning of BPD and the literature on psychoanalytic psychopathology. These results have several important clinical implications.

Perspectives de l'inactivation des pathogènes: quand la fiction dépasse la réalité [Back to the future: a fiction dealing with pathogen inactivation of blood products].

AIM OF THE PAPER: Arouse the reflection with a fiction having a scientific appearance, presenting a late and unexpected complication of the universal inactivation of pathogens. CONCLUSION: Such a fiction story opens the debate on a series of fundamental questions that could be addressed during the paradigm shift that is expected by introducing universal pathogen inactivation of blood products.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.