TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.

Neural crest cell survival is dependent on Rho kinase and is required for development of the mid face in mouse embryos.

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia.

TNF-alpha-dependent loss of IKKbeta-deficient myeloid progenitors triggers a cytokine loop culminating in granulocytosis.

Loss of IκB kinase (IKK) β-dependent NF-κB signaling in hematopoietic cells is associated with increased granulopoiesis. Here we identify a regulatory cytokine loop that causes neutrophilia in Ikkβ-deficient mice. TNF-α-dependent apoptosis of myeloid progenitor cells leads to the release of IL-1β, which promotes Th17 polarization of peripheral CD4(+) T cells. Although the elevation of IL-17 and the consecutive induction of granulocyte colony-stimulating factor compensate for the loss of myeloid progenitor cells, the facilitated induction of Th17 cells renders Ikkβ-deficient animals more susceptible to the development of experimental autoimmune encephalitis. These results unravel so far unanticipated direct and indirect functions for IKKβ in myeloid progenitor survival and maintenance of innate and Th17 immunity and raise concerns about long-term IKKβ inhibition in IL-17-mediated diseases.

Comparison of topography- and aperture-dependent motion compensation algorithms for airborne SAR

This letter presents a comparison between threeFourier-based motion compensation (MoCo) algorithms forairborne synthetic aperture radar (SAR) systems. These algorithmscircumvent the limitations of conventional MoCo, namelythe assumption of a reference height and the beam-center approximation.All these approaches rely on the inherent time–frequencyrelation in SAR systems but exploit it differently, with the consequentdifferences in accuracy and computational burden. Aftera brief overview of the three approaches, the performance ofeach algorithm is analyzed with respect to azimuthal topographyaccommodation, angle accommodation, and maximum frequencyof track deviations with which the algorithm can cope. Also, ananalysis on the computational complexity is presented. Quantitativeresults are shown using real data acquired by the ExperimentalSAR system of the German Aerospace Center (DLR).

Condition-dependent genetic benefits of extrapair fertilization in female blue tits Cyanistes caeruleus.

In many socially monogamous birds, both partners perform extrapair copulations (EPC). As this behaviour potentially inflicts direct costs on females, they are currently hypothesized to search for genetic benefits for descendants, either as 'good' or 'complementary' genes. Although these hypotheses have found some support, several studies failed to find any beneficial consequence of EPC, and whether this behaviour is adaptive to females is subject to discussion. Here, we test these two hypotheses in a natural population of blue tits by accounting for the effect of most parameters known to potentially affect extrapair fertilization. Results suggest that female body mass affected the type of extrapair genetic benefits obtained. Heavy females obtained extrapair fertilizations when their social male was of low quality (as reflected by sexual display) and produced larger extrapair than within-pair chicks. Lean females obtained extrapair fertilizations when their social mate was genetically similar, thereby producing more heterozygous extrapair chicks. Our results suggest that mating patterns may be condition-dependent.

Oxidants positively or negatively regulate nuclear factor kappaB in a context-dependent manner.

Redox-based mechanisms play critical roles in the regulation of multiple cellular functions. NF-kappaB, a master regulator of inflammation, is an inducible transcription factor generally considered to be redox-sensitive, but the modes of interactions between oxidant stress and NF-kappaB are incompletely defined. Here, we show that oxidants can either amplify or suppress NF-kappaB activation in vitro by interfering both with positive and negative signals in the NF-kappaB pathway. NF-kappaB activation was evaluated in lung A549 epithelial cells stimulated with tumor necrosis factor alpha (TNFalpha), either alone or in combination with various oxidant species, including hydrogen peroxide or peroxynitrite. Exposure to oxidants after TNFalpha stimulation produced a robust and long lasting hyperactivation of NF-kappaB by preventing resynthesis of the NF-kappaB inhibitor IkappaB, thereby abrogating the major negative feedback loop of NF-kappaB. This effect was related to continuous activation of inhibitor of kappaB kinase (IKK), due to persistent IKK phosphorylation consecutive to oxidant-mediated inactivation of protein phosphatase 2A. In contrast, exposure to oxidants before TNFalpha stimulation impaired IKK phosphorylation and activation, leading to complete prevention of NF-kappaB activation. Comparable effects were obtained when interleukin-1beta was used instead of TNFalpha as the NF-kappaB activator. This study demonstrates that the influence of oxidants on NF-kappaB is entirely context-dependent, and that the final outcome (activation versus inhibition) depends on a balanced inhibition of protein phosphatase 2A and IKK by oxidant species. Our findings provide a new conceptual framework to understand the role of oxidant stress during inflammatory processes.

Expression of the transcription factor NFAT2 in human neutrophils: IgE-dependent, Ca2+- and calcineurin-mediated NFAT2 activation.

NFAT (nuclear factors of activated T cells) proteins constitute a family of transcription factors involved in mediating signal transduction. The presence of NFAT isoforms has been described in all cell types of the immune system, with the exception of neutrophils. In the present work we report for the first time the expression in human neutrophils of NFAT2 mRNA and protein. We also report that specific antigens were able to promote NFAT2 protein translocation to the nucleus, an effect that was mimicked by the treatment of neutrophils with anti-immunoglobulin E (anti-IgE) or anti-Fcepsilon-receptor antibodies. Antigens, anti-IgE and anti-FcepsilonRs also increased Ca2+ release and the intracellular activity of calcineurin, which was able to interact physically with NFAT2, in parallel to eliciting an enhanced NFAT2 DNA-binding activity. In addition, specific chemical inhibitors of the NFAT pathway, such as cyclosporin A and VIVIT peptide, abolished antigen and anti-IgE-induced cyclooxygenase-2 (COX2) gene upregulation and prostaglandin (PGE(2)) release, suggesting that this process is through NFAT. Our results provide evidence that NFAT2 is constitutively expressed in human neutrophils, and after IgE-dependent activation operates as a transcription factor in the modulation of genes, such as COX2, during allergic inflammation.

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Age-related changes in sleep in inbred mice are genotype dependent.

Aging produces major changes in sleep structure and intensity which might be linked to cognitive impairment in the elderly. In this study, the genetic contribution to age-related changes in sleep was assessed in three inbred mouse strains of various ages. Baseline sleep and the response to 6 hours sleep deprivation (SD) achieved by gentle handling were quantified in young, middle-aged, and older male mice using electroencephalography. Total sleep time initially increased with age but then decreased in the oldest group mainly due to changes in sleep duration during the active phase. The effect of age on electroencephalographic (EEG) delta power depends on genotype and sleep pressure level with SD increasing the age-related differences. The strong effect of age upon the spectral profile of the different behavioral states was modulated by genetic background. Overall, our results suggest that sleep pressure can modulate the effect of age, that most sleep variables do not monotonically change with age in contrast to previous reports in humans and other species, and that genetic factors have a major impact on the aging processes affecting sleep.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

Maternal and paternal contributions to pathogen resistance dependent on development stage in a whitefish (Salmonidae)

It is often assumed that maternal and paternal contributions to offspring phenotype change over the lifetime of an individual. However, studies on parental effects typically suffer from the problems that heritabilities and maternal environmental effects are difficult to separate, and that both may depend on environmental factors and developmental stage. In order to experimentally disentangle maternal from paternal contributions and the likely effects of developmental stage from ecological effects, we sampled a natural population of the whitefish Coregonus palaea, used gametes for full-factorial in vitro fertilizations, raised over 10000 of the resulting offspring singly at controlled conditions, and exposed them at different points during embryonic development to one of two strains of Pseudomonas fluorescens that differed in their virulence characteristics (only one caused mortality, while both delayed hatching and reduced growth). Vulnerability to infection increased markedly over embryo development. This change coincided with a distinct shift in the importance of maternal to additive genetic effects on survival. Timing of exposure also affected the variance components for hatching time and larval length, but in a less consistent direction than the variance components for mortality. No significant genetic variation was found for any reaction norms across time points of exposure, indicating a uniformity among genotypes in how susceptibility changed over development. Phenotypes were also typically correlated across time points, which could constrain the evolution of the reaction norms. Our experiment demonstrates that the relative maternal and paternal contributions to susceptibility to an infection, and hence the evolutionary potential to respond to pathogen-induced selection, depends not only on the kind of pathogenic stress but also on the timing of the challenge.