Ultrastructural data on the life cycle of the parasite, Perkinsus atlanticus (Apicomplexa), on the clam, Ruditapes phillipinarum, in the Mediterranean

An Apicomplexan Perkinsus species has been found parasitizing the clam Ruditapes philippinarum (= Tapes semidecussatus) collected on the Mediterranean coast in the region of the Ebro Delta (Tarragona, Spain). Light and transmission electron microscopy were used to study different stages of this parasite during zoosporulation induced by incubation in thioglycollate medium and seawater. During incubation the trophozoites began zoosporulation, which originated prezoosporangia and zoosporangia at different developmental stages. Successive cytokinesis and nucleokinesis gave rise to prezoospores, which became elongate and differentiated in biflagellated zoospores. The latter presented large mitochondria and an apical complex formed by a conoid, polar ring, micronemes, rhophtries and subpellicular microtubules. The zoosporangium wall showed some typical lamosomes and a discharge tube developed in early phases of incubation. Ultrastructural data were compared with the only four species of the genus Perkinsus previously described. The morphological data, the host and the geographic proximity suggest that the species located on the Mediterranean coast was Perkinsus atlanticus.

NS4B Self-Interaction through Conserved C-Terminal Elements Is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.

Role of hepatocyte wounding on " Plasmodium " liver-stage development and survival

RESUME La première étape primordiale au cycle de vie du Plasmodium dans un hôte mammifère est l'invasion des hepatocytes par des sporozoites. L'infection finale des hepatocytes est précédée de la traversée de plusieurs cellules hôtes, rompant les membranes plasmiques et ayant comme résultat la sécrétion des facteurs cytotoliques dans le micro-environnement. Ce matériel endogène libéré est fortement stimulant/immunogène et peut servir de signal de danger initiant des réponses distinctes dans diverses cellules. De nos jours, le caractère essentiel et salutaire de la migration des sporozoites comme étape d'infection du Plasmodium est vivement controversée. Ainsi, notre étude a visé à caractériser l'effet de l'interaction du parasite avec ses cellules hôtes d'un point de vue immunologique. En particulier, nous avons voulu évaluer l'effet de la perte de matériel cellulaire pendant l'infection de Plasmodium sur les hepatocytes primaires de souris et sur des cultures cellulaires HepG2. Nous avons observé que les facteurs cytotoxiques dérivés des cellules endommagés activent NF-κB - un important régulateur de réponse inflammatoires -dans des cellules voisines des cellules endommagés, qui sont des cellules hôtes potentielles pour l'infection finale du parasite. Cette activation de NF-κB s'est produite peu de temps après l'infection et a mené in vitro et in vivo à une réduction d'infection de façon dépendante du temps, un effet qui a pu être compensé par l'addition de BAY11-7082, un inhibiteur spécifique de NF-κB. De plus, aucune activation de NF-κB avec des parasites SPECT-/-, incapables de traverser les hepatocytes, n'a été observée. Nous avons montré parla suite que l'activation de NF-κB induit l'expression de l'enzyme iNOS dans les hepatocytes, qui est responsable d'une diminution des hepatocytes infectés. En outre, les hepatocytes primaires des souris MyD88-/- n'ont montré ni activation de NF-κB, ni expression d'iNOS lors de l'infection, ce qui suggère la participation des membres de famille du Toll/IL-1 récepteur dans la reconnaissance des facteurs cytosoxiques. En effet, le manque de MyD88 a augmenté significativement l'infection in vitro et in vivo. D'autre part, un rôle bénéfique pour l'activation de NF-κB a été évalué. Les cellules infectées étaient plus résistantes contre l'apoptose induite par Fas (CD95/Apo-1) que les cellules non infectées ou les cellules infectées dans lesquelles NF-κB a été bloqué par BAY11-7082 in vitro. Paradoxalement, l'expression d'iNOS contribue à la protection des cellules infectées contre l'apoptose pax Fas, puisque le traitement avec l'inhibiteur spécifique SMT (S-methylisothiourea) a rendu les cellules infectées plus susceptibles à l'apoptose. Un effet bénéfique additionnel pour le parasite est que la plupart des cellules hôtes traversées présentent des peptides du parasite aux cellules T cytotoxiques spécifiques et peuvent donc réorienter la réaction immune spécifique sur les cellules non infectées. Nous montrons que les cellules hôtes endommagés par la migration du parasite induit l'inflammation, qui limite l'ampleur de l'infection. D'autre part, nos données soutiennent que la survie du parasite Plasmodium dans le foie est assurée par une augmentation de la résistance des hepatocytes contre l'apoptose. SUMMARY The first obligatory step of the Plasmodium life cycle in the mammalian host is the invasion of hepatocytes by sporozoites. Final hepatocyte infection involves the penetration of several host cells, whose plasma membranes are ruptured in the process, resulting in the release of cytosolic factors into the microenvironment. This released endogenous material is highly stimulatory / immunogenic and can serve as a danger signal initiating distinct responses in various cells. To date, it is highly controversial whether sporozoite migration through hepatocytes is an essential and beneficial step for Plasmodium infection. Thus, our study aimed at characterizing the effect of the interaction of the parasite with its host cells from an immunological point of view In particular, we wanted to evaluate the effect of cell material leakage during Plasmodium infection on cultured mouse primary hepatocytes and HepG2 cells. We observed that wounded cell-derived cytosolic factors activate NF-κB - a main regulator of host inflammatory responses - in cells bordering wounded cells, which are potential host cells for final parasite infection. This activation of NF-κB occurred shortly after infection and led to a reduction of infection load in a time dependent manner in vitro and in viva, an effect that could be reverted by addition of the specific NF-κB inhibitor BAY11-7082. In addition, no NF-κB activation was observed when SPECT-/- parasites, which are devoid of hepatocyte traversing properties, were used. We provide further evidence that NF-κB activation causes the induction of inducible nitric oxide synthase (iNOS) expression in hepatocytes, and this is, in turn, responsible for a decrease in Plasmodium-infected hepatocytes. Furthermore, primary hepatocytes from MyD88-/- mice showed no NF-κB activation and iNOS expression upon infection, suggesting a role of the Toll/IL-1 receptor family members in sensing cytosolic factors. Indeed, lack of MyD88 significantly increased infection in vitro and in vivo. In a further complementary series of experiments, we assessed a possible beneficial role for the activation of NF-κB. Infected cells were more resistant to Fas (CD95/Apo-1)-mediated apoptosis than uninfected cells or infected cells in which NF-κB was blocked by BAYl1-7082 in vitro. Paradoxically, iNOS expression contributes to the protection of infected cells from Fas-induced apoptosis, since treatment with the specific iNOS inhibitor SMT (S-Methylisothiourea Sulfate) rendered the infected cells more susceptible to apoptosis. An additional beneficial effect of host cell traversal for the parasite is the fact that mainly traversed cells present parasite-derived peptides to specific cytotoxic T cells and therefore may redirect the specific immune response to uninfected cells. In summary, we have shown that host cells wounded by parasite migration induce inflammation, which limits the extent of parasite infection. In addition, our data support the notion that survival of Plasmodium parasites in the liver is mediated by increasing the resistance of hepatocytes to Fas-induced apoptosis.

Accurate calculations of intermolecular interaction energies using explicitly correlated coupled cluster wave functions and a dispersion-weighted MP2 method.

Explicitly correlated coupled-cluster calculations of intermolecular interaction energies for the S22 benchmark set of Jurecka, Sponer, Cerny, and Hobza (Chem. Phys. Phys. Chem. 2006, 8, 1985) are presented. Results obtained with the recently proposed CCSD(T)-F12a method and augmented double-zeta basis sets are found to be in very close agreement with basis set extrapolated conventional CCSD(T) results. Furthermore, we propose a dispersion-weighted MP2 (DW-MP2) approximation that combines the good accuracy of MP2 for complexes with predominately electrostatic bonding and SCS-MP2 for dispersion-dominated ones. The MP2-F12 and SCS-MP2-F12 correlation energies are weighted by a switching function that depends on the relative HF and correlation contributions to the interaction energy. For the S22 set, this yields a mean absolute deviation of 0.2 kcal/mol from the CCSD(T)-F12a results. The method, which allows obtaining accurate results at low cost, is also tested for a number of dimers that are not in the training set.

Leukocyte-endothelial cell interaction is necessary for photodynamic therapy induced vascular permeabilization.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) affects vascular barrier function and thus increases vessel permeability. This phenomenon may be exploited to facilitate targeted drug delivery and may lead to a new clinical application of photodynamic therapy. Here, we investigate the role of leukocyte recruitment for PDT-induced vascular permeabilization. STUDY DESIGN/MATERIAL AND METHODS: Fluorescein isothiocyanate dextran (FITC-D, 2,000 kDa) was injected intravenously 120 minutes after focal PDT on striated muscle in nude mice bearing dorsal skinfold chambers (Visudyne® 800 µg/kg, fluence rate 300 mW/cm2 , light dose of 200 J/cm2). Leukocyte interaction with endothelial cells was inhibited by antibodies functionally blocking adhesion molecules ("MABS-PDT" group, n = 5); control animals had PDT but no antibody injection (group "PDT", n = 7). By intravital microscopy, we monitored leukocyte rolling and sticking in real-time before, 90 and 180 minutes after PDT. The extravasation of FITC-D from striated muscle vessels into the interstitial space was determined in vivo during 45 minutes to assess treatment-induced alterations of vascular permeability. RESULTS: PDT significantly increased the recruitment of leukocytes and enhanced the leakage of FITC-D. Neutralization of adhesion molecules before PDT suppressed the rolling of leukocytes along the venular endothelium and significantly reduced the extravasation of FITC-D as compared to control animals (156 ± 27 vs. 11 ± 2 (mean ± SEM, number of WBC/30 seconds mm vessel circumference; P < 0.05) at 90 minutes after PDT and 194 ± 21 vs. 14 ± 4 at 180 minutes after PDT). In contrast, leukocyte sticking was not downregulated by the antibody treatment. CONCLUSION: Leukocyte recruitment plays an essential role in the permeability-enhancing effect of PDT.

Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the contribution of tumor necrosis factor is minimal.

Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.

Nouveaux traitements de l'hépatite C: aspects pharmacologiques et potentiel d'interaction [New hepatitis C treatments: pharmacological considerations and potential for drug interactions].

Progress in the understanding of the hepatitis C virus life cycle allowed the development of new, very promising antiviral therapies. Although these new drugs have a favourable profile in terms of efficacy, tolerance and interaction potential, their prescription in the setting of comedication and impaired renal or hepatic function remains a challenge. Here, we provide a summary of pharmacological considerations, focusing on sofosbuvir, simeprevir and daclatasvir. A better understanding of their metabolic pathways and transporters may help the prescriber to identify and manage drug interactions especially in patients under immunosuppressive or anti-HIV therapy. Recommendations for the prescription of these drugs in specific situations are also discussed.

DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-kappaB.

Detection of viral nucleic acids is central to antiviral immunity. Recently, DAI/ZBP1 (DNA-dependent activator of IRFs/Z-DNA binding protein 1) was identified as a cytoplasmic DNA sensor and shown to activate the interferon regulatory factor (IRF) and nuclear factor-kappa B (NF-kappaB) transcription factors, leading to type-I interferon production. DAI-induced IRF activation depends on TANK-binding kinase 1 (TBK1), whereas signalling pathways and molecular components involved in NF-kappaB activation remain elusive. Here, we report the identification of two receptor-interacting protein (RIP) homotypic interaction motifs (RHIMs) in the DAI protein sequence, and show that these domains relay DAI-induced NF-kappaB signals through the recruitment of the RHIM-containing kinases RIP1 and RIP3. We show that knockdown of not only RIP1, but also RIP3 affects DAI-induced NF-kappaB activation. Importantly, RIP recruitment to DAI is inhibited by the RHIM-containing murine cytomegalovirus (MCMV) protein M45. These findings delineate the DAI signalling pathway to NF-kappaB and suggest a possible new immune modulation strategy of the MCMV.

Interaction of prechilling, temperature, osmotic stress, and light in Picea abies seed germination

Summary

Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity : a role for microglia

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

The evolutionary host switches of Polychromophilus: a multi-gene phylogeny of the bat malaria genus suggests a second invasion of mammals by a haemosporidian parasite.

BACKGROUND: The majority of Haemosporida species infect birds or reptiles, but many important genera, including Plasmodium, infect mammals. Dipteran vectors shared by avian, reptilian and mammalian Haemosporida, suggest multiple invasions of Mammalia during haemosporidian evolution; yet, phylogenetic analyses have detected only a single invasion event. Until now, several important mammal-infecting genera have been absent in these analyses. This study focuses on the evolutionary origin of Polychromophilus, a unique malaria genus that only infects bats (Microchiroptera) and is transmitted by bat flies (Nycteribiidae). METHODS: Two species of Polychromophilus were obtained from wild bats caught in Switzerland. These were molecularly characterized using four genes (asl, clpc, coI, cytb) from the three different genomes (nucleus, apicoplast, mitochondrion). These data were then combined with data of 60 taxa of Haemosporida available in GenBank. Bayesian inference, maximum likelihood and a range of rooting methods were used to test specific hypotheses concerning the phylogenetic relationships between Polychromophilus and the other haemosporidian genera. RESULTS: The Polychromophilus melanipherus and Polychromophilus murinus samples show genetically distinct patterns and group according to species. The Bayesian tree topology suggests that the monophyletic clade of Polychromophilus falls within the avian/saurian clade of Plasmodium and directed hypothesis testing confirms the Plasmodium origin. CONCLUSION: Polychromophilus' ancestor was most likely a bird- or reptile-infecting Plasmodium before it switched to bats. The invasion of mammals as hosts has, therefore, not been a unique event in the evolutionary history of Haemosporida, despite the suspected costs of adapting to a new host. This was, moreover, accompanied by a switch in dipteran host.

Altitudinal variation in haemosporidian parasite distribution in great tit populations.

BACKGROUND: One of the major issues concerning disease ecology and conservation is knowledge of the factors that influence the distribution of parasites and consequently disease outbreaks. This study aimed to investigate avian haemosporidian composition and the distribution of these parasites in three altitudinally separated great tit (Parus major) populations in western Switzerland over a three-year period. The objectives were to determine the lineage diversity of parasites occuring across the study populations and to investigate whether altitudinal gradients govern the distribution of haemosporidian parasites by lineage. METHODS: In this study molecular approaches (PCR and sequencing) were used to detect avian blood parasites (Plasmodium sp., Haemoproteus sp. and Leucocytozoon sp.) in populations of adult great tits caught on their nests during three consecutive breeding seasons. RESULTS: High levels of parasite prevalence (88-96%) were found across all of the study populations with no significant altitude effect. Altitude did, however, govern the distribution of parasites belonging to different genera, with Plasmodium parasites being more prevalent at lower altitudes, Leucocytozoon parasites more at high altitude and Haemoproteus parasite prevalence increasing with altitude. A total of 27 haemosporidian parasite lineages were recorded across all study sites, with diversity showing a positive correlation to altitude. Parasites belonging to lineage SGS1 (P. relictum) and PARUS4 and PARUS19 (Leucocytozoon sp.) dominated lower altitudes. SW2 (P. polare) was the second most prevalent lineage of parasite detected overall and these parasites were responsible for 68% of infections at intermediate altitude, but were only documented at this one study site. CONCLUSIONS: Avian haemosporidian parasites are not homogeneously distributed across host populations, but differ by altitude. This difference is most probably brought about by environmental factors influencing vector prevalence and distribution. The high occurrence of co-infection by different genera of parasites might have pronounced effects on host fitness and should consequently be investigated more rigorously.

Interaction analysis and psychology: A dialogical perspective

Interaction analysis is not a prerogative of any discipline in social sciences. It has its own history within each disciplinary field and is related to specific research objects. From the standpoint of psychology, this article first draws upon a distinction between factorial and dialogical conceptions of interaction. It then briefly presents the basis of a dialogical approach in psychology and focuses upon four basic assumptions. Each of them is examined on a theoretical and on a methodological level with a leading question: to what extent is it possible to develop analytical tools that are fully coherent with dialogical assumptions? The conclusion stresses the difficulty of developing methodological tools that are fully consistent with dialogical assumptions and argues that there is an unavoidable tension between accounting for the complexity of an interaction and using methodological tools which necessarily "monologise" this complexity.