Clusterin Neutralizes the Inflammatory and Cytotoxic Properties of Extracellular Histones in Sepsis.

Rationale: Extracellular histones, released into the surrounding environment during extensive cell death, promote inflammation and cell death, and these deleterious roles have been well documented in sepsis. Clusterin (CLU) is a ubiquitous extracellular protein that chaperones misfolded proteins and promotes their removal. Objectives: We investigated whether CLU could protect against the deleterious properties of histones. Methods: We assessed CLU and histone expression in patients with sepsis and evaluated the protective role of CLU against histones in in vitro assays and in vivo models of experimental sepsis. Measurements and Main Results: We show that CLU binds to circulating histones and reduces their inflammatory, thrombotic, and cytotoxic properties. We observed that plasma CLU levels decreased in patients with sepsis and that the decrease was greater and more durable in nonsurvivors than in survivors. Accordingly, CLU deficiency was associated with increased mortality in mouse models of sepsis and endotoxemia. Finally, CLU supplementation improved mouse survival in a sepsis model. Conclusions: This study identifies CLU as a central endogenous histone-neutralizing molecule and suggests that, in pathologies with extensive cell death, CLU supplementation may improve disease tolerance and host survival.

IL-26 inhibits hepatitis C virus replication in hepatocytes.

BACKGROUND & AIMS: Interleukin-26 (IL-26) is a proinflammatory cytokine that has properties atypical for a cytokine, such as direct antibacterial activity and DNA-binding capacity. We previously observed an accumulation of IL-26 in fibrotic and inflammatory lesions in the livers of patients with chronic HCV infection and showed that infiltrating CD3+ lymphocytes were the principal source of IL-26. Surprisingly, IL-26 was also detected in the cytoplasm of hepatocytes from HCV-infected patients, even though these cells do not produce IL-26, even when infected with HCV. Based on this observation and possible interactions between IL-26 and nucleic acids, we investigated the possibility that IL-26 controlled HCV infection independently of the immune system. METHODS: We evaluated the ability of IL-26 to interfere with HCV replication in hepatocytes and investigated the mechanisms by which IL-26 exerts its antiviral activity. RESULTS: We showed that IL-26 penetrated HCV-infected hepatocytes, where it interacted directly with HCV double-stranded RNA replication intermediates, thereby inhibiting viral replication. IL-26 interfered with viral RNA-dependent RNA polymerase activity, preventing the de novo synthesis of viral genomic single-stranded RNA. CONCLUSIONS: These findings reveal a new role for IL-26 in direct protection against HCV infection, independently of the immune system, and increase our understanding of the antiviral defense mechanisms controlling HCV infection. Future studies should evaluate the possible use of IL-26 for treating other chronic disorders caused by RNA viruses, for which few treatments are currently available, or emerging RNA viruses. LAY SUMMARY: This study sheds new light on the body's arsenal for controlling hepatitis C virus (HCV) infection and identifies interleukin-26 (IL-26) as an antiviral molecule capable of blocking HCV replication. IL-26, which has unique biochemical and structural characteristics, penetrates infected hepatocytes and interacts directly with viral RNA, thereby blocking viral replication. IL-26 is, therefore, a new player in antiviral defenses, operating independently of the immune system. It is of considerable potential interest for treating HCV infection and other chronic disorders caused by RNA viruses for which few treatments are currently available, and for combating emerging RNA viruses.

An invasive prey provides long-lasting silver spoon effects for an endangered predator.

The natal environment can have long-term fitness consequences for individuals, particularly via 'silver spoon' or 'environmental matching' effects. Invasive species could alter natal effects on native species by changing species interactions, but this potential remains unknown. Using 17 years of data on 2588 individuals across the entire US breeding range of the endangered snail kite (Rostrhamus sociabilis), a wetland raptor that feeds entirely on Pomacea snails, we tested for silver spoon and environmental matching effects on survival and movement and whether the invasion of a non-native snail may alter outcomes. We found support for silver spoon effects, not environmental matching, on survival that operated through body condition at fledging, explained by hydrology in the natal wetland. When non-native snails were present at the natal site, kites were in better condition, individual condition was less sensitive to hydrology, and kites fledged across a wider range of hydrologic conditions, leading to higher survival that persisted for at least 10 years. Movement between wetlands was driven by the current (adult) environment, and birds born in both invaded and uninvaded wetlands preferred to occupy invaded wetlands post-fledging. These results illustrate that species invasions may profoundly impact the role of natal environments on native species.

Towards a unified framework for connectivity that disentangles movement and mortality in space and time.

Predicting connectivity, or how landscapes alter movement, is essential for understanding the scope for species persistence with environmental change. Although it is well known that movement is risky, connectivity modelling often conflates behavioural responses to the matrix through which animals disperse with mortality risk. We derive new connectivity models using random walk theory, based on the concept of spatial absorbing Markov chains. These models decompose the role of matrix on movement behaviour and mortality risk, can incorporate species distribution to predict the amount of flow, and provide both short- and long-term analytical solutions for multiple connectivity metrics. We validate the framework using data on movement of an insect herbivore in 15 experimental landscapes. Our results demonstrate that disentangling the roles of movement behaviour and mortality risk is fundamental to accurately interpreting landscape connectivity, and that spatial absorbing Markov chains provide a generalisable and powerful framework with which to do so.

IL-26 Confers Proinflammatory Properties to Extracellular DNA.

In physiological conditions, self-DNA released by dying cells is not detected by intracellular DNA sensors. In chronic inflammatory disorders, unabated inflammation has been associated with a break in innate immune tolerance to self-DNA. However, extracellular DNA has to complex with DNA-binding molecules to gain access to intracellular DNA sensors. IL-26 is a member of the IL-10 cytokine family, overexpressed in numerous chronic inflammatory diseases, in which biological activity remains unclear. We demonstrate in this study that IL-26 binds to genomic DNA, mitochondrial DNA, and neutrophil extracellular traps, and shuttles them in the cytosol of human myeloid cells. As a consequence, IL-26 allows extracellular DNA to trigger proinflammatory cytokine secretion by monocytes, in a STING- and inflammasome-dependent manner. Supporting these biological properties, IL-10-based modeling predicts two DNA-binding domains, two amphipathic helices, and an in-plane membrane anchor in IL-26, which are structural features of cationic amphipathic cell-penetrating peptides. In line with these properties, patients with active autoantibody-associated vasculitis, a chronic relapsing autoimmune inflammatory disease associated with extensive cell death, exhibit high levels of both circulating IL-26 and IL-26-DNA complexes. Moreover, in patients with crescentic glomerulonephritis, IL-26 is expressed by renal arterial smooth muscle cells and deposits in necrotizing lesions. Accordingly, human primary smooth cells secrete IL-26 in response to proinflammatory cytokines. In conclusion, IL-26 is a unique cationic protein more similar to a soluble pattern recognition receptor than to conventional cytokines. IL-26 expressed in inflammatory lesions confers proinflammatory properties to DNA released by dying cells, setting up a positive amplification loop between extensive cell death and unabated inflammation.

MICA variant promotes allosensitization after kidney transplantation.

MHC class I-related chain A (MICA) antigens are surface glycoproteins strongly implicated in innate immunity, and the MICA gene is highly polymorphic. Clinical observations suggest a role for donor MICA antigens expressed on transplant endothelial cells in the alloimmune response, but the effect of MICA genotype is not well understood. Here, we investigated the immunologic effect of the A5.1 mutation, related to the common MICA*008 allele. Compared with wild-type endothelial cells (ECs), homozygosity for MICA A5.1 associated with an endothelial phenotype characterized by 7- to 10-fold higher levels of MICA mRNA and MICA proteins at the cell surface, as well as exclusive release in exosomes instead of enzymatic cleavage. Mechanistically, we did not detect quantitative changes in regulatory microRNAs. Functionally, A5.1 ECs enhanced NKG2D interaction and natural killer cell activation, promoting NKG2D-dependent lysis of ECs. In kidney transplant recipients, polyreactive anti-MICA sera bound preferentially to ECs from MICA A5.1 donors, suggesting that MICA*008(A5.1) molecules are the preferential antigenic determinants on ECs of grafts. Furthermore, the incidence of MICA A5.1 mismatch revealed a statistically significant association between donor MICA A5.1 and both anti-MICA sensitization and increased proteinuria in kidney recipients. Taken together, these results identify the A5.1 mutation as an immunodominant factor and a potential risk factor for transplant survival.

Foray movements are common and vary with natal habitat for a highly mobile bird.

Understanding dispersal is central to interpreting the effects of climate change, habitat loss and habitat fragmentation, and species invasions. Prior to dispersal, animals may gather information about the surrounding landscape via forays, or systematic, short-duration looping movements away from and back to the original location. Despite theory emphasizing that forays can be beneficial for dispersing organisms and that such behaviors are predicted to be common, relatively little is known about forays in wild populations. Theory predicts that individuals that use forays may delay dispersal and such behaviors should increase survival, yet empirical tests of these predictions remain scarce. We tested these predictions in a natural system using the critically endangered snail kite (Rostrhaumus sociabilis), a wetland-dependent raptor. We GPS tracked 104 snail kites from fledging through emigration from the natal site across their breeding range to understand the demographic consequences of movement. We found that forays were common (82.7% of individuals tracked), and natal habitat played an important role in the initiation, execution, and outcome of foray behavior. The effect of foraying on survival was indirect, where forayers emigrated later than non-forayers, and individuals that emigrated later had the highest survival. Poor hydrological conditions in the natal environment were especially important for eliciting forays. Finally, females responded more strongly to natal hydrology than males, making more forays and significantly longer, more distant trips. These results emphasize the fundamental role of natal habitat for determining behavioral patterns, strengthen links between individual movement decisions and their demographic consequences, and provide an important behavioral focal point for interpreting movement tracks that would not otherwise be captured by conventional movement models.

MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer.

Generation of tumor-antigen specific CD4(+) T-helper (T(H)) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4(+) T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T(H) lines. We isolated phenotypically defined CD4(+) T-cell subpopulations from circulating lymphocytes of DR52b(+) healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO(119-143), autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO(119-143) tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer(+) cells by co-staining with TCR variable ß chain (BV) specific antibodies. We isolated ESO-tetramer(+) cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T(H) lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer(+) T(H) lines from conventional CD4(+)CD25(-) naïve and central memory populations, but not from effector memory populations or CD4(+)CD25(+) Treg. In vitro primed T(H) lines recognized ESO with affinities comparable to ESO-tetramer(+) cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal T(H) lines from non-immune individuals. This is an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.

Progressive multifocal leukoencephalopathy after durvalumab treatment for acute myeloid leukemia: A consequence of an immune reconstitution inflammatory syndrome?

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system resulting from the reactivation of the John Cunningham virus (JCV). PML occurs almost exclusively during profound immune suppression but it can also be observed in immunocompromised subjects as part of an inflammatory immune reconstitution syndrome (IRIS) in patients receiving antiviral therapy. We report a case of PML in a 61-year-old patient with acute myeloid leukemia who had developed after discontinuation of durvalumab (anti-PD-L1) therapy initiated after multiple treatments. Results suggest that PML may result from two nonexclusive mechanisms: (i) an inhibition of the protective response of JCV-specific T cells as a consequence of the blockade of the PD1-PDL1 pathway, associated with a lack of compensatory expression of other inhibitory receptors by T cells and (ii) a neuroinflammatory response (PML-IRIS) that may have contributed to virus reactivation.

IL-26, a Cytokine With Roles in Extracellular DNA-Induced Inflammation and Microbial Defense.

Interleukin 26 (IL-26) is the most recently identified member of the IL-20 cytokine subfamily, and is a novel mediator of inflammation overexpressed in activated or transformed T cells. Novel properties have recently been assigned to IL-26, owing to its non-conventional cationic, and amphipathic features. IL-26 binds to DNA released from damaged cells and, as a carrier molecule for extracellular DNA, links DNA to inflammation. This observation suggests that IL-26 may act both as a driver and an effector of inflammation, leading to the establishment of a deleterious amplification loop and, ultimately, sustained inflammation. Thus, IL-26 emerges as an important mediator in local immunity/inflammation. The dysregulated expression and extracellular DNA carrier capacity of IL-26 may have profound consequences for the chronicity of inflammation. IL-26 also exhibits direct antimicrobial properties. This review summarizes recent advances on the biology of IL-26 and discusses its roles as a novel kinocidin.

Hydroxychloroquine partially prevents endothelial dysfunction induced by anti-beta-2-GPI antibodies in an in vivo mouse model of antiphospholipid syndrome.

BACKGROUND: Antiphospholipid syndrome is associated with endothelial dysfunction, which leads to thrombosis and early atheroma. Given that hydroxychloroquine has anti-thrombotic properties in lupus, we hypothesized that it could reduce endothelial dysfunction in an animal model of antiphospholipid syndrome. We evaluated the effect of hydroxychloroquine in preventing endothelial dysfunction in a mouse model of antiphospholipid syndrome. METHODS: Antiphospholipid syndrome was induced by an injection of monoclonal anti-beta-2-GPI antibodies. Vascular reactivity was evaluated in mesenteric resistance arteries isolated from mice 3 weeks (APL3W) after receiving a single injection of anti-beta-2-GPI antibodies and after 3 weeks of daily oral hydroxychloroquine treatment (HCQ3W) compared to control mice (CT3W). We evaluated endothelial dysfunction by measuring acetylcholine-mediated vasodilation. A pharmacological approach was used to evaluate NO synthase uncoupling (tetrahydrobiopterin) and the generation of reactive oxygen species (Tempol). RESULTS: Impaired acetylcholine-mediated dilation was evidenced in mice 3 weeks after anti-beta-2-GPI antibodies injection compared to CT3W, by reduced maximal dilation (p<0.0001) and sensitivity (pKd) (p = 0.01) to acetylcholine. Hydroxychloroquine improved acetylcholine-dependent dilation, on pKd (p = 0.02) but not maximal capacity compared to untreated mice. The addition of tetrahydrobiopterin (p = 0.02) and/or Tempol (p = 0.0008) improved acetylcholine-mediated dilation in APL3W but not in HCQ3W. CONCLUSIONS: We demonstrated that endothelial dysfunction in mouse resistance arteries persisted at 3 weeks after a single injection of monoclonal anti-beta-2-GPI antibodies, and that hydroxychloroquine improved endothelium-dependent dilation at 3 weeks, through improvement of NO synthase coupling and oxidative stress reduction.

Thoracic and cutaneous sarcoid-like reaction associated with anti-PD-1 therapy: longitudinal monitoring of PD-1 and PD-L1 expression after stopping treatment.

BACKGROUND: Immune checkpoint inhibitors (ICI) target T cell inhibitory pathways that are responsible for cancer tolerance by down-modulating immune functions. ICI have revolutionized patients care with lung cancer. Nevertheless, restoring endogenous antitumor T-cell responses can induce immune related adverse events, such as sarcoidosis. CASE PRESENTATION: We report here the first case of a thoracic and cutaneous sarcoid-like reaction in a patient with a relapsing unresectable non-small cell lung cancer (NSCLC) treated with nivolumab, an anti-PD-1 mAb. The expression of PD-1 and its ligands, PD-L1 and PD-L2, was assessed by flow cytometry on peripheral blood mononuclear cells (PBMC) and compared to patients who had discontinued nivolumab therapy without having developed any immune related adverse events. PD-L1 expression was transiently increased on B cells, T cells and monocytes, whereas PD-L2 expression was not modulated. PD-1 was transiently undetectable when PD-L1 was maximal, before returning to basal level. Sarcoidosis spontaneously resolved, without corticotherapy. CONCLUSION: This case sheds the light on a complex regulation of PD-L1 expression in vivo on PBMC after nivolumab arrest and triggers the question of monitoring the expression of immune checkpoint on immune cells during and after treatment with ICI.

Dynamic oceanography determines fine scale foraging behavior of Masked Boobies in the Gulf of Mexico.

During breeding, foraging marine birds are under biological, geographic, and temporal constraints. These contraints require foraging birds to efficiently process environmental cues derived from physical habitat features that occur at nested spatial scales. Mesoscale oceanography in particular may change rapidly within and between breeding seasons, and findings from well-studied systems that relate oceanography to seabird foraging may transfer poorly to regions with substantially different oceanographic conditions. Our objective was to examine foraging behavior of a pan-tropical seabird, the Masked Booby (Sula dactylatra), in the understudied Caribbean province, a moderately productive region driven by highly dynamic currents and fronts. We tracked 135 individuals with GPS units during May 2013, November 2013, and December 2014 at a regionally important breeding colony in the southern Gulf of Mexico. We measured foraging behavior using characteristics of foraging trips and used area restricted search as a proxy for foraging events. Among individual attributes, nest stage contributed to differences in foraging behavior whereas sex did not. Birds searched for prey at nested hierarchical scales ranging from 200 m-35 km. Large-scale coastal and shelf-slope fronts shifted position between sampling periods and overlapped geographically with overall foraging locations. At small scales (at the prey patch level), the specific relationship between environmental variables and foraging behavior was highly variable among individuals but general patterns emerged. Sea surface height anomaly and velocity of water were the strongest predictors of area restricted search behavior in random forest models, a finding that is consistent with the characterization of the Gulf of Mexico as an energetic system strongly influenced by currents and eddies. Our data may be combined with tracking efforts in the Caribbean province and across tropical regions to advance understanding of seabird sensing of the environment and serve as a baseline for anthropogenic based threats such as development, pollution, and commercial fisheries.

Anti-pentraxin antibodies in autoimmune systemic diseases: Focus on anti-pentraxin-3 autoantibodies.

Pentraxins are soluble pattern recognition molecules implicated not only in the opsonization and removal of microorganisms but also in the clearance of modified self (i.e., dying cells). Pentraxins can be classified into short pentraxins (C-reactive protein and serum amyloid-P component) and long pentraxins with pentraxin-3 (PTX3), the prototypic one of the latter. Pentraxins are involved in various physiological or pathophysiological processes such as inflammation and autoimmunity. A breakdown in immune tolerance to pentraxins has been reported in various autoimmune diseases. In the present review, we analyzed the current knowledge concerning anti-pentraxin antibodies, with a special focus on anti-PTX3 autoantibodies.

Comparison of two enzymatic immunoassays, high resolution mass spectrometry method and radioimmunoassay for the quantification of human plasma histamine.

Histamine (HA) is one of the main immediate mediators involved in allergic reactions. HA plasma concentration is well correlated with the severity of vascular and respiratory signs of anaphylaxis. Consequently, plasma quantification of HA is useful to comfort the diagnosis of anaphylaxis. Currently, radioimmunoassay (RIA) is the gold standard method to quantify HA due to its high sensitivity, but it is time consuming, implicates specific formations and cautions for technicians, and produces hazardous radioactive wastes. The aim of this study was to compare two enzymatic immunoassays (EIA) and one in-house liquid chromatography high-resolution mass spectrometry method (LC-HRMS) with the gold standard method for HA quantification in plasma samples of patients suspected of anaphylaxis reactions. Ninety-two plasma samples were tested with the 4 methods (RIA, 2 EIA and LC-HRMS) for HA quantification. Fifty-eight samples displayed HA concentrations above the positive cut-off of 10nM evaluated by RIA, including 18 highly positive samples (>100 nM). This study shows that Immunotech(®) EIA and LC-HRMS concentrations were highly correlated with RIA values, in particular for samples with a HA concentration around the positive cut-off. In our hands, plasma concentrations obtained with the Demeditec Diagnostics(®) EIA correlated less with results obtained by RIA, and an underestimation of plasma HA levels led to a lack of sensitivity. In conclusion, this study demonstrates that Immunotech(®) EIA and LC-HRMS method could be used instead of RIA to assess plasma HA in human diagnostic use.

Low Serum Complement C3 Levels at Diagnosis of Renal ANCA-Associated Vasculitis Is Associated with Poor Prognosis.

BACKGROUND: Recent studies have demonstrated the key role of the complement alternative pathway (cAP) in the pathophysiology of experimental ANCA-associated vasculitis (AAV). However, in human AAV the role of cAP has not been extensively explored. In the present work, we analysed circulating serum C3 levels measured at AAV onset and their relation to outcomes. METHODS: We conducted a retrospective observational cohort study including 45 consecutive patients with AAV diagnosed between 2000 and 2014 with serum C3 measurement at diagnosis, before immunosuppressive treatment initiation. Two groups were defined according to the median serum C3 level value: the low C3 group (C3<120 mg/dL) and the high C3 level group (C3≥120 mg/dL). Patient and renal survivals, association between C3 level and renal pathology were analysed. RESULTS: Serum complement C3 concentration remained in the normal range [78-184 mg/dL]. Compared with the high C3 level, the patients in the low C3 level group had lower complement C4 concentrations (P = 0.008) and lower eGFR (P = 0.002) at diagnosis. The low C3 level group had poorer patient and death-censored renal survivals, compared with the high C3 level group (P = 0.047 and P = 0.001, respectively). We observed a significant negative correlation between C3 levels and the percentage of glomeruli affected by cellular crescent (P = 0.017, r = -0.407). According to the Berden et al renal histologic classification, patients in the crescentic/mixed category had low C3 levels more frequently (P<0.01). Interestingly, we observed that when patients with the crescentic/mixed histologic form were analysed according to C3 level, long term renal survival was significantly greater in the high C3 level group than in the low C3 level group (100% vs 40.7% at 6 years, p = 0.046). No relationship between serum C4 and renal outcome was observed. CONCLUSION: A Low C3 serum level in AAV patients at diagnosis is associated with worse long-term patient and renal survival.

Receptor activating NF-κB ligand (RANKL) is a constitutive intracellular protein in resting human basophils and is strongly induced on their surface by interleukin 3.

Receptor activating NF-κB ligand (RANKL) is a member of the TNF superfamily that plays a pivotal role in bone homeostasis as being the major osteoclastogenesis factor. RANKL also has pleiotropic effects in the immune system in which it is expressed by activated T and B cells and some innate lymphoid cells. RANKL-RANK interactions mediate lymph node organogenesis and immunoregulatory functions in autoimmune disease and carcinogenesis as well as cross talk between the immune system and bone. In this study, we show that basophils were the strongest RANKL mRNA-expressing cells amongst major leukocyte subsets in human blood. RANKL was preformed as an intracellular protein in resting basophils and was rapidly and strongly expressed on their surface upon stimulation with IL-3, but not other stimuli. This expression was stable for at least 6 days. Activated basophils could also release soluble RANKL in small quantities upon interaction with DCs or monocytes. In the blood, basophils were the sole cells to express membrane RANKL in response to IL-3. This study indicates that basophils should be considered as new players in the pleiotropic and complex RANKL-RANK interaction system and suggests a role for RANKL in the interaction between basophils and immune cells in inflammatory allergic tissues and secondary lymphoid organs.

Emergency testing for antineutrophil cytoplasmic antibodies combined with a dialog-based policy between clinician and biologist: effectiveness for the diagnosis of ANCA-associated vasculitis.

A prompt immunosuppressive treatment initiation is crucial in ANCA-associated vasculitis (AAV) to minimize organ injury. The aim of the present work was to analyze the accuracy of emergency ANCA screening to identify rapidly patients with AAV. In our Institution, emergency ANCA screening is based on a telephone call between a Clinician and a Biologist. Indirect immunofluorescence (IIF) for ANCA detection was performed using a commercial kit (Euroimmun(®) Granulocyte Mosaic 12). Positive serums for c- or p-ANCA at IIF are subsequently screened for antigenic specificity (MPO or PR3) by an immunodot technique (immunodot, D-Tek(®).) Positive samples with atypical c- or p-ANCA pattern at IIF are subsequently screened for antigenic specificity by ELISA. Data were retrieved from patients' medical records and confronted to emergency ANCA screening results. Between 2005 and 2012, 114 patients were screened. IIF was positive in 27.2% of patients, but c-/p-ANCA anti-MPO/-PR3 was detected in 13.2% of patients. The sensibility and specificity of IIF combined with immunodot for newly diagnosed AAV were 83.3 and 100%, respectively. Ten patients were newly diagnosed with AAV. In these patients, a specific AAV treatment was initiated less than 24 h following ANCA screening. Emergency ANCA screening based on a clinical gating policy was relevant to identify patients with AAV diagnosis, and was associated with a rapid treatment initiation.

A novel piggyBac transposon inducible expression system identifies a role for AKT signalling in primordial germ cell migration.

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.