Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild-yet chronic-neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1-but not pertussis toxin, which affects Gi protein-dependent responses-abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis

Vernalization affects DNA methylation of the VRN-A1 gene in hexaploid wheat

International audienc

Uneven genotypic diversity of Escherichia coli in fecal sources limits the performance of a library-dependent method of microbial source tracking on the southwestern French Atlantic coast

International audienceTo develop a library-dependent method of tracking fecal sources of contamination of beaches on the Atlantic coast of southwestern France, a library of 6368 Escherichia coli isolates was constructed from samples of feces, from 40 known human or animal sources collected in the vicinity of Arcachon Bay in 2010, and in French Basque Country, Landes, and Beam, between 2017 and 2018. Different schemes of source identification were tested: use of the complete or filtered reference library; characterization of the isolates by genotypic or proteomic profiling based on ERIC-PCR or MALDI-TOF mass spectrometry, respectively; isolate by isolate assignment using either classifiers based on the Pearson similarity or SVM (support vector machine). With the exception of one source identification scheme, which was discarded since it used self-assignment, all tested schemes resulted in low rates of correct classification (15%). The heterogeneous coverage of E. coli genotypic diversity between sources and the uneven distribution of E. coli genotypes in the library likely explain the difficulties encountered in identifying the sources of fecal contamination. Shannon diversity index of sources ranged from 0 for several wildlife species sampled once to 3.03 for sewage treatment plant effluents sampled on various occasions, showing discrepancies between sources. The uneven genotypic composition of the library was attested by the value of the Pielou index (0.54), the high proportion of nondiscriminatory genotypes (>91% of the isolates), and the very low proportion of discriminatory genotypes (<3%). Since efforts made to constitute such a library are not affordable for routine analyses, the results question the relevance of developing such a method for identifying sources of fecal contamination on such a coastline

Human bronchial epithelium orchestrates dendritic cell activation in severe asthma

International audienceThe innate immune response is impaired in asthma, with increased epithelial release of C-X-C motif chemokine ligand (CXCL) 8, interleukin (IL)-33 and thymic stromal lymphopoietin (TSLP). We hypothesised that dendritic cells might modulate the hyperresponsive epithelium in severe asthma. For this purpose, we investigated epithelial-dendritic crosstalk in normal and diseased conditions, and because ultrafine particulate matter may affect asthmatic airways, we investigated its impact on this crosstalk. Air-liquid interface cultures of human bronchial epithelial cells (HBEC) of control subjects (cHBEC) or severe asthma patients (saHBEC) were co-cultured with monocyte-derived dendritic cells (moDC). Increased release of CXCL8, TSLP and IL-33 from saHBEC contrasted with cHBEC producing CXCL10 and CCL2. Regarding moDC activation, saHBEC co-cultures induced only upregulation of CD86 expression, while cHBEC yielded full moDC maturation with HLA-DR, CD80, CD86 and CD40 upregulation. Particulate matter stimulation of HBEC had no effect on cHBEC but stimulated CXCL8 and IL-33 release in saHBEC. Particulate matter impaired epithelium signalling (TSLP, IL-33 and CXCL8) in saHBEC co-cultures despite C-C chemokine ligand 2 induction. Crosstalk between HBEC and moDC can be established in vitro, driving a T1-type response with cHBEC and a T2-type response with saHBEC. Normal or asthmatic status of HBEC differentially shapes the epithelial-dendritic responses. We conclude that control moDC cannot rescue the hyperresponsive airway epithelium of severe asthmatics

Streamlining basophil activation testing to enable assay miniaturization and automation of sample preparation

Background: Numerous studies have demonstrated the capabilities of the basophil activation test (BAT) but various parameters such as a lack of standardization and a time consuming and labor intensive workflow continue to hinder the field to fully leverage the capabilities of this technique. When pediatric patients have to be considered, an additional limitation is related to blood volume consumption. Objectives: This work aimed at developing and characterizing a simplified and standardized whole-blood based BAT prototype procedure and at further assessing the feasibility of automating and miniaturizing the developed assay into a 96 well plate format. Methods: A dry and room temperature stable reagent technology was used to simplify and standardize BAT. Under optimized conditions, EDTA anticoagulated whole blood samples of non-allergic and allergic donors ( < 24 h old) together with calcium containing buffer were added to ready-to-use dry reagent tubes or 96 well plates (negative controls, positive controls and allergen tests) containing a 5 color compensation-free antibody panel (CD45-KrO/CD3-PC7/CRTH2-A647/CD203c-PE/CD63-PB). Upon mixing and incubation at 37 degrees C for 15 min, erythrocytes were lysed and samples were analyzed by flow cytometry without further washing steps. While it is important to precisely control the incubation time to minimize the assay variability, herein, a 15 min incubation time was chosen as it provides a suitable compromise for both the magnitude of basophil activation and the quality of the staining. A Biomek NXP robotic platform (Beckman Coulter) was used for automation and both CD203c and CD63 levels were monitored to characterize basophil reactivity. Results: This streamlined BAT protocol is no-wash, compensation free and only requires 4 pipetting steps to be completed. The assessment of assay performance characteristics showed wide applicability, satisfactory repeatability and a high degree of standardization as demonstrated by very low infra-assay and inter-operator variabilities (CVs < 10%). Leveraging these technical foundations, it was then proven that this new BAT procedure can easily be transposed into the 96 well plate format, thereby benefiting from a miniaturized format and full automation capabilities. When considering 8 dilution points to characterize the ex vivo basophil reactivity of a given whole blood sample, we found that as little as 51.1.L of blood per point could be used. Conclusions: A whole blood based and simplified procedure for BAT is proposed. It relies on a dry antibody formulation technology and requires only a few manual steps to be completed. This procedure can also be transposed in a 96 well plate format, fully automated and miniaturized, when sample volume reduction, throughput increase or unattended sample preparation is required

Role of environmental fluctuations and microbial diversity in degradation of hydrocarbons in contaminated sludge.

cited By 12International audienceLittle is known about microbial communities involved in hydrocarbon degradation, whether it be their structural and functional diversity or their response to environmental constraints such as oxygen fluctuation. Here, current knowledge of the impact of diversity and redox oscillations upon ecosystem processes is reviewed. In addition, we present the main conclusions of our studies in this field. Oxic/anoxic oscillations had a strong impact upon bacterial community structures, influencing their ability to degrade hydrocarbons and their capacity to reduce hydrocarbon toxicity. Furthermore, a decrease in functional diversity has a strong impact on pollutant degradation

Hydrocarbon degradation in coastal muddy areas and anoxic ecosystems (DHYVA project): Role of bacterial mechanisms and bioturbation effects on the biodisponibility of organic pollutants

International audienc

Response to commentary by Drs. Poncet and Sénéchal

International audienceWe thank Drs. Poncet and Sénéchal for their interest and critical reading of our paper. We are well aware of their pioneering work on molecular aspects of cypress pollinosis. However, the focus of our paper was rather on epidemiological, clinical and diagnostic features of peach allergy and not towards molecular pollen determinants. We hereby provide answers to direct questions and notions made by Drs. Poncet and Sénéchal. 1. In regard to literature references, we cited those we found relevant for the scope and purpose of the paper and none of the three reviewers suggested citation of additional publications. To our knowledge, the paper by Hugues et al (2006) was indeed the first to report an association between cypress and peach allergy, and it is cited as reference 39 in our paper 1. However, contrary to the assertion by Poncet and Sénéchal, the authors of that paper did not identify a pollen homologue of Pru p 7, which was first reported as an allergen by Tuppo et al in 2013 2 but instead made the notion "Because both allergenic extracts include a 45 kDa-allergen, it should be the shared allergen." Cup a 1, a major allergen in cypress pollen, has a molecular weight of 43 kDa. Experimental data from the Poncet team are currently available for BP14 and snakin-1, neither of which have been officially recognized and named as allergens by the WHO/IUIS Allergen Nomenclature SubCommittee (www.aller gen.org, accessed May 4 2019). Other papers cited by Poncet et al are either replies or reviews. We prefer citation of original, peer-reviewed research. However, the review on cypress pollinosis is also cited in our paper as ref 41. 3 2. This case report of one patient with discordant FABER IgE and BAT results would have brought little if any further information to the reader. In our hands, the FABER test displays highly sensitive detection of IgE to Pru p 7. 3. As noted above, BP14 has not been officially recognized as an allergen. 4. Snakin-1 is out of the scope of our publication. 5. Recombinant Pru p 7 was biochemically and immunologically characterized as described in section 2.6 4 and additionally by circular dichroism spectroscopy, but, given the focus of the paper, we did not consider it relevant to show and elaborate on such data, nor was there space available. It was also not suggested by any of the three reviewers. However, the BAT results shown in Table S3 demonstrate a similar functional potency of natural and recombinant Pru p 7 which suggests an authentic folding of the recombinant protein. o We do not agree that assessment of anti-microbial activity of recombinant Pru p 7 and several of the other specifics mentioned would be necessary to validate the association between cypress pollen allergy and peach allergy as suggested (but not yet done in their publications) by Drs Poncet and Sénéchal. o The cypress species used was Cupressus sempervirens. o The pollen was extracted and clarified by standard methods. We did not consider total protein concentration to be informative in relation to the purpose of the experiment but chose instead to determine the potency of the extract by titrated inhibition of IgE binding to Pru p 7, as described in section 3.5. That potency determination guided the choice of inhibitor concentration in the single-point inhibitions shown in figure 5A. 15% (w/v) means a concentration corresponding to 15 g of pollen (dry weight) per 100 mL of liquid, a manner of expressing concentrations also used by Poncet et al in their papers. o The specificity of inhibition with the pollen extract was ensured by a complete lack of inhibition of binding of dog dander specific IgE to dog dander ImmunoCAP (e5) and is further indicated by the lack of significant inhibition in some samples as shown in figure 5A. Had the inhibitory effect of the cypress pollen extract been due to unspecific blockade of IgE, no such results would have been obtained. We hope that our response will provide sufficient clarity and explanation to the questions raised by Drs. Poncet and Sénéchal