Seroreactivity against specific L5P antigen from <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> in children at risk for T1D

Aims/Hypothesis: Although numerous environmental agents have been investigated over the years as possible triggers of type 1 diabetes (T1D), its causes remain unclear. We have already demonstrated an increased prevalence of antibodies against peptides derived from Mycobacterium avuim subsp. paratuberculosis (MAP) homologous to human zinc transporter 8 protein (ZnT8) and proinsulin in Italian subjects at risk for or affected by T1D. In this study, we compared titers of the previously detected antibodies with seroreactivity to MAP lipopentapetide (L5P) that recently emerged as a strong immunogenic component able to specifically distinguish MAP from other mycobacteria. Methods: Plasma of 32 children and youth at risk for T1D including follow-up samples and 42 age-matched healthy controls (HC) recruited at the Tor Vergata University Hospital in Rome was analyzed by indirect ELISA for the presence of antibodies against MAP-derived epitopes MAP3865c133–141, MAP3865c125-133, MAP2404c70-85 and MAP1,4αgbp157-173 along with their ZnT8 and proinsulin homologs. The data were analyzed through two-tailed Mann-Whitney U test and relation between variables was determined by principal component analysis. Results: Responses to L5P were not detectable in subjects whose initial seroreactivity to MAP peptides and their human homologs was lost in follow-up samples, whereas anti-L5P antibodies appeared constantly in individuals with a stable immunity against MAP antigens. The overall coincidence in positivity to L5P and the four MAP epitopes both in children at risk for T1D and HC exceeded 90%. Conclusions: MAP-derived homologs may cross-react with ZnT8 and proinsulin peptides inducing immune responses at a young age in subjects predisposed for T1D. Thus, L5P may have a diagnostic value to immediately indicate the presence of anti-MAP seroreactivity when evaluation of a more complex antibody status is not required. Almost complete coincidence in responses to both types of antigens lends support to the involvement of MAP in T1D

Removing critical gaps in chemical test methods by developing new assays for the identification of thyroid hormone system-disrupting chemicals—the athena project

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood–brain and blood–placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation

Outcome after allogeneic stem cell transplantation with haploidentical versus HLA-matched donors in patients with higher-risk MDS.

peer reviewedAllogeneic hematopoietic stem cell transplantation remains the best curative option for higher-risk myelodysplastic syndrome. The presence of monosomal karyotype and/or complex karyotype abnormalities predicts inferior survival after allo-SCT in MDS patients. Haploidentical allo-SCT has been increasingly used in acute leukemia (AL) and has similar results as using HLA-matched donors, but data on higher-risk MDS is sparse. We compared outcomes in 266 patients with higher-risk MDS after HLA-matched sibling donor (MSD, n = 79), HLA-matched unrelated donor (MUD, n = 139) and HLA haploidentical donor (HID, n = 48) from 2010 to 2019. Median donor age differed between the three groups (p < 0.001). The overall survival was significantly different between the three groups with a better OS observed in the MUD group (p = 0.014). This observation could be explained by a higher progression-free survival with MUD (p = 0.014). The cumulative incidence of grade 2-4 acute GvHD was significantly higher in the HID group (p = 0.051). However, in multivariable analysis, patients transplanted using an HID had comparable mortality to patients transplanted using a MUD (subdistribution hazard ratio [sHR]: 0.58 [0.32-1.07]; p = 0.080) and a MSD ([sHR]: 0.56 [0.28-1.11]; p = 0.094). MUD do not remain a significant positive predictor of survival, suggesting that beyond the donor-recipient HLA matching, the donor age might impact recipient outcome

Efficient monitoring of enzymatic conjugation reaction by surface-enhanced laser desorption/ionization time of flight mass spectrometry for process optimization.

International audienceEfficient analysis of bioconjugation reactions is one the most challenging task for optimizing and eventually achieving the reproducible production of large amount of conjugates. In particular, the complexity of some reaction mixtures precludes the use of most of the existing methods, because of the presence of large amounts of contaminants. As an alternative method, we used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for monitoring an in vitro enzymatic transglycosylation of N-acetylgalactosamine (GalNAc) residues to a recombinant mucin protein MUC6. For this reaction, catalyzed by the uridine 5'-diphospho-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), we used either a recombinant ppGalNAc-T1 or a mixture of ppGalNAc-Ts contained in MCF7 tumor cell extracts. In the present study, we show that SELDI-TOF MS offers unique advantages over the traditional methodologies. It is a rapid, accurate, sensitive, reproducible, and very convenient analytical method for monitoring the course of a bioconjugation, even in heterogeneous samples such as cell extracts. SELDI-TOF MS proved very useful for optimizing the reaction parameters of the transglycosylation and for achieving the large scale preparation of Tn antigen-glycosylated mucins for antitumor immunotherapy applications

Combined genomic, machine learning and biochemical approaches for the characterization of lipopeptides from Mycobacterium avium subsp. paratuberculosis.

National audienceIntroduction and objectives: Mycobacterium avium subsp. paratuberculosis (Map), the agent of paratuberculosis or Johne's disease, is responsible for considerable economic losses in the dairy industry worldwide. This animal pathogenic mycobacterium is a Nontuberculous Mycobacteria (NTM) of the Mycobacterium avium complex (MAC). Genomic studies initiated in 2016, and the recent complete genomes obtained, have confirmed the clonal nature of this subspecies, which is divided into three distinct genetic lineages designated CII (for Bovine strains), SI and SIII (for Ovine strains). Our previous studies have established that, unlike other mycobacteria of the avium complex, Map does not produce glycopeptidolipids on the surface of its cell wall, but rather sugar-free lipopeptide antigens. Molecular and genetic characterization of these antigens, which are synthesized by Non-Ribosomal Peptide Synthases (NRPS), has shown that they are unique to Map and may differ between genetic lineages. In this study, we have characterized previously unidentified antigens.Materials and methods: Genomic sequencing of new Map strains was carried out in Illumina and PacBio to obtain complete genomes of these strains. Genomic analyses using machine learning approaches were used to identify new NRPS and make metabolite predictions. Chemical synthesis produced predicted antigens to guide formal identification and characterization of native antigens by mass spectrometry (MALDI-TOF, HPLC-MS) and NMR analyses.Results, discussion and conclusion: Genomic and biochemical analyses have enabled us to characterize a new lipopeptide antigen in Map. Our results add to our knowledge of the lipopeptides produced in Map, and open up new prospects for the development of serological diagnostics based on synthetic molecules, in the same way as current diagnostics using a cell extract that is not specific to Map and difficult to produce

Antigenic tripeptides derived from Mycobacterium avium subsp. paratuberculosis S-type strains, derivatives and uses thereof

Ce brevet a été déposé le 20 décembre 2016 sous le numéro PCT/FR2016/053590The present invention is directed to the diagnosis, prevention and treatment of diseases resulting from infections by Mycobacterium avium subsp. paratuberculosis.The present invention is directed to an isolated synthetic tripeptide of formula H-D-Phe- N-Methyl- L-Val-L-Ala-OMe (SEQ ID NO :1), or a derivative thereof, and to the corresponding lipotripeptides, which are specific to Mycobacterium avium subsp. paratuberculosis (Map) S-type strain, as well as derivatives and conjugates thereof. The invention also concerns the use of these antigens in different methods and tests for detecting Map infection, especially by detecting humoral response and cell mediated response of infected animals. The invention is also directed to a genetic signature of Map and a mass spectrometry and NMR spectroscopy signature of Map presence or infection

The Pta-AckA pathway controlling acetyl phosphate levels and the phosphorylation state of the DegU orphan response regulator both play a role in regulating Listeria monocytogenes motility and chemotaxis

International audienceDegU is considered to be an orphan response regulator in Listeria monocytogenes since the gene encoding the cognate histidine kinase DegS is absent from the genome. We have previously shown that DegU is involved in motility, chemotaxis and biofilm formation and contributes to L. monocytogenes virulence. Here, we have investigated the role of DegU phosphorylation in Listeria and shown that DegS of Bacillus subtilis can phosphorylate DegU of L. monocytogenes in vitro. We introduced the B. subtilis degS gene into L. monocytogenes, and showed that this leads to highly increased expression of motility and chemotaxis genes, in a DegU-dependent fashion. We inactivated the predicted phosphorylation site of DegU by replacing aspartate residue 55 with asparagine and showed that this modified protein (DegU(D55N)) is no longer phosphorylated by DegS in vitro. We show that although the unphosphorylated form of DegU retains much of its activity in vivo, expression of motility and chemotaxis genes is lowered in the degU(D55N) mutant. We also show that the small-molecular-weight metabolite acetyl phosphate is an efficient phosphodonor for DegU in vitro and our evidence suggests this is also true in vivo. Indeed, a L. monocytogenesDeltaptaDeltaackA mutant that can no longer synthesize acetyl phosphate was found to be strongly affected in chemotaxis and motility gene expression and biofilm formation. Our findings suggest that phosphorylation by acetyl phosphate could play an important role in modulating DegU activity in vivo, linking its phosphorylation state to the metabolic status of L. monocytogenes

L5P a specific lipopeptide of <em>Mycobacterium avium</em> subp. <em>paratuberculosis</em>: feature and innovative diagnosis applications

National audienceJohne’s disease or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a major worldwide health problem of domestic and wild animals. All control programmes developed until now against this large epizootie have failed due to the lack of sensitive and specific diagnostic assays and to the lack of an efficient vaccine [1]. The research of new cell wall antigens represent an original alternative to protein antigens typically used in commercial sero-diagnostic tests. Many non-tuberculous mycobacteria such as Mycobacterium avium subsp. avium (Mav) synthesize abundant glycopeptidolipids (GPLs). These surfacelocated GPLs are involved in pathogenicity by interfering with the host immune system. In Mav, GPLs consist of a lipopeptide core composed of a tetrapeptide O-linked to mono- and oligo-saccharides. The biosynthesis pathway of the simplest GPLs is now relatively well understood and involves probably more than fifteen genes [2]. Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs. To provide a genomic basis for the synthesis of this compound, the published genome sequence of Map was explored using in silico methods. Even though Map produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of Mav. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav and is in agreement with the amino acid content of the L5P. To circumvent the problems of challenging native purification, L5P was chemically synthesized which allows the large-scale production of pure L5P [3]. We further showed that L5P is the target for a highly specific humoral response involving IgM, IgG1 and IgG2 antibodies in Map-infected animals, and that the major epitopes of the L5P are localized in the peptidyl moiety of the molecule. We also showed L5P is the target for a specific humoral response in a subset of human patients with Crohn disease (CD). The L5P, a molecular signature of Map, will open the way to an innovative ELISA diagnosis based on a novel synthetic antigen to unambiguously distinguish Map from Mav, M. bovis or other environmental mycobacteria in sera from livestock or from CD patients [4]. T cell assay and direct detection using specific monoclonal antibodies available should complete diagnosis of Map from blood, milk, and lesions

L5P a specific lipopeptide of[i] Mycobacterium avium[/i] subp. [i]Paratuberculosis[/i]: feature and innovative diagnosis applications

Session III : Innate and adaptive responses to microbes. The beneficial and harmful aspectsJohne’s disease or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a major worldwide health problem of domestic and wild animals. All control programmes developed until now against this large epizootie have failed due to the lack of sensitive and specific diagnostic assays and to the lack of efficient vaccine. The research of new cell wall antigens represent an original alternative to protein antigens typically used in commercial sero-diagnostic tests. Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs produced by many non-tuberculous mycobacteria such as Mycobacterium avium subsp. avium (Mav). To provide a genomic basis for the synthesis of this compound, the genome of Map was explored. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as Mav and is in agreement with the amino acid content of the L5P. To circumvent the problems of challenging native purification, L5P was chemically synthesized which allows the large-scale production of pure L5P. We further showed that L5P is the target for a highly specific humoral and that the major epitopes of the L5P are localized in the peptidyl moiety of the molecule. We also showed L5P is the target for a specific humoral response in a subset of human patients with Crohn disease (CD). The L5P, a molecular signature of Map, will open the way to an innovative ELISA diagnosis to unambiguously distinguish Map from Mav, M. bovis or other environmental mycobacteria in sera from livestock or from CD patients. T cell assay and direct detection using specific monoclonal antibodies available should complete diagnosis of Map from blood, milk, and lesions