Untargeted metabolomics as a hypothesis-generation tool in plant protection product discovery: Highlighting the potential of trehalose and glycerol metabolism of fungal conidiospores as novel targets.

INTRODUCTION: The production of high quality and safe food represents a main priority for the agri-food sector in the effort to sustain the exponentially growing human population. Nonetheless, there are major challenges that require the discovery of new, alternative, and improved plant protection products (PPPs). Focusing on fungal plant pathogens, the dissection of mechanisms that are essential for their survival provides insights that could be exploited towards the achievement of the aforementioned aim. In this context, the germination of fungal spores, which are essential structures for their dispersal, survival, and pathogenesis, represents a target of high potential for PPPs. To the best of our knowledge, no PPPs that target the germination of fungal spores currently exist. OBJECTIVES: Within this context, we have mined for changes in the metabolite profiles of the model fungus Aspergillus nidulans FGSC A4 conidiospores during germination, in an effort to discover key metabolites and reactions that could potentially become targets of PPPs. METHODS: Untargeted GC/EI-TOF/MS metabolomics and multivariate analyses were employed to monitor time-resolved changes in the metabolomes of germinating A. nidulans conidiospores. RESULTS: Analyses revealed that trehalose hydrolysis plays a pivotal role in conidiospore germination and highlighted the osmoregulating role of the sugar alcohols, glycerol, and mannitol. CONCLUSION: The ineffectiveness to introduce active ingredients that exhibit new mode(s)-of-action as fungicides, dictates the urge for the discovery of PPPs, which could be exploited to combat major plant protection issues. Based on the crucial role of trehalose hydrolysis in conidiospore dormancy breakage, and the subsequent involvement of glycerol in their germination, it is plausible to suggest their biosynthesis pathways as potential novel targets for the next-generation antifungal PPPs. Our study confirmed the applicability of untargeted metabolomics as a hypothesis-generation tool in PPPs' research and discovery.

Multiple nucleobase transporters contribute to boscalid sensitivity in Aspergillus nidulans.

The development of fungicide-resistant fungal populations represents a major challenge for the agrochemical and agri-food sectors, which threatens food supply and security. The issue becomes complex for fungi that cause quantitative and qualitative losses due to mycotoxin biosynthesis. Nonetheless, currently, the molecular details underlying fungicide action and fungal resistance mechanisms are partially known. Here, we have investigated whether plasma membrane transporters contribute to specific fungicide uptake in the model fungus Aspergillus nidulans. Independent physiological tests and toxicity screening of selected fungicides provided evidence that the antifungal activity of Succinate Dehydrogenase Inhibitors (SDHIs) is associated with the expression of several nucleobase-related transporters. In particular, it was shown that a strain genetically inactivated in all seven nucleobase-related transporters is resistant to the fungicide boscalid, whereas none of the single null mutants exhibited significant resistance level. By constructing and testing isogenic strains that over-express each one of the seven transporters, we confirmed that five of them, namely, UapC, AzgA, FycB, CntA, and FurA, contribute to boscalid uptake. Additionally, by employing metabolomics we have examined the effect of boscalid on the metabolism of isogenic strains expressing or genetically lacking boscalid-related nucleobase transporters. The results confirmed the involvement of specific nucleobase transporters in fungicide uptake, leading to the discovery of corresponding metabolites-biomarkers. This work is the first report on the involvement of specific transporters in fungicide uptake and toxicity and their impact on fungal metabolism regulation and results might be further exploited towards the deeper understanding of fungal resistance to fungicides.

Peritoneal cavity regulatory B cells (B10 cells) modulate IFN-γ+CD4+ T cell numbers during colitis development in mice.

The spleen regulatory B cell subset with the functional capacity to express IL-10 (B10 cells) modulates both immune responses and autoimmune disease severity. However, the peritoneal cavity also contains relatively high frequencies of functionally defined IL-10-competent B10 cells. In this study, peritoneal cavity B10 cells shared similar cell surface phenotypes with their spleen counterparts. However, peritoneal cavity B10 cells were 10-fold more frequent among B cells than occurred within the spleen, intestinal tract, or mesenteric lymph nodes and were present at higher proportions among the phenotypically defined peritoneal B1a > B1b > B2 cell subpopulations. The development or localization of B10 cells within the peritoneal cavity was not dependent on the presence of commensal microbiota, T cells, IL-10 or B10 cell IL-10 production, or differences between their fetal liver or adult bone marrow progenitor cell origins. The BCR repertoire of peritoneal cavity B10 cells was diverse, as occurs in the spleen, and predominantly included germline-encoded VH and VL regions commonly found in either the conventional or B1 B cell compartments. Thereby, the capacity to produce IL-10 appears to be an intrinsic functional property acquired by clonally diverse B cells. Importantly, IL-10 production by peritoneal cavity B cells significantly reduced disease severity in spontaneous and induced models of colitis by regulating neutrophil infiltration, colitogenic CD4(+) T cell activation, and proinflammatory cytokine production during colitis onset. Thus, the numerically small B10 cell subset within the peritoneal cavity has regulatory function and is important for maintaining homeostasis within gastrointestinal tissues and the immune system.

Successful management of refractory pediatric-onset complex aphthosis with lenalidomide.

Clinicians are frequently confronted with patients presenting with oral aphthous ulcers or orogenital aphthae. Patients with complex aphthosis are characterized by the nearly constant presence of more than 3 oral aphthous ulcers or recurrent orogenital aphthae but do not satisfy the criteria for Behçet disease. We report a severe case of pediatric-onset complex aphthosis with poor response and/or significant toxicity to first-line medications. Lenalidomide, a second-generation immunomodulatory drug, induced a complete disease remission within few days of therapy. The patient has been treated for nearly 3 years with significant subjective and clinical improvement and no adverse effects. Thereby, lenalidomide may represent a well-tolerated and effective medication for patients with complex aphthosis who are intolerant or not responsive to first-line agents. Nevertheless, in view of the rare but serious potential adverse effects of lenalidomide such as teratogenicity and cancer, a clinical trial is necessary to assess the true risk-to-benefit ratio for the use of lenalidomide in patients with complex aphthosis.

Metabolomic and Genomic Approach to Study Defense Induction by Nesidiocoris tenuis against Tuta absoluta and Tetranychus urticae in Tomato Plants.

The phytophagy of the predator Nesidiocoris tenuis (Hemiptera: Miridae) can trigger defense responses in tomato plants against pests, such as two spotted spider mite Tetranychus urticae (Acari: Tetranychidae) and South American leaf miner Tuta absoluta (Lepidoptera: Gelechiidae). The expression of genes governing Jasmonic Acid (JA) biosynthesis pathway and fluctuations in the levels of underlying metabolites have been rarely studied in mirid-infested plants. In the present study, fifteen 3rd instar nymphs of N.tenuis were caged on each top and lower leaf of tomato plants for 4 d to induce plant defense; after this period the predators were removed. With regard to T. absoluta, oviposition preference; larval period; and pupal weight were significantly reduced in N. tenuis-punctured plants. T. urticae adults exhibited a significantly higher escape tendency and reduced survival on punctured plants. Metabolomics confirmed such observations revealing substantial differences between N. tenuis-punctured and unpunctured (control) plants. Metabolites directly associated with the activation of the JA defense pathway, such as the precursor α-linolenic acid, had increased concentrations. The expression of the defense-related genes PI-II, MYC2, VSP2, and HEL was increased in the top leaves and only VSP2 and MBP2 in the lower leaves; interestingly, in the middle (unpunctured) leaves VSP2, HEL, and MBP2 were also upregulated, indicating systemic signaling. Collectively, phytophagy of N. tenuis caused adverse effects on T. absoluta and T. urticae, whereas the multi-omics approach (phenomics, metabolomics, and genomics) offered valuable insights into the nature of the plant defense responses and provided useful evidence for future applications in integrated pest management, plausibly resulting in the reduction in the required pesticide volumes.

Abatacept in the treatment of localized scleroderma: A pediatric case series and systematic literature review.

BACKGROUND: Localized scleroderma (LS) is a rare chronic immune-mediated skin condition of unknown etiology characterized by an inflammatory response in the skin and subcutaneous tissues resulting in collagen deposition and subsequent fibrosis. There is no cure for LS. No therapies have been licensed specifically for the treatment of LS and the clinical management of the disease remains largely empirical. Abatacept, a recombinant fusion protein interfering with the T-cell costimulatory pathway, has been reported to be effective in adult cases of LS. We report the successful use of abatacept in a juvenile localized scleroderma (jLS) cohort and conduct a systematic literature review to evaluate the evidence supporting the use of abatacept in the treatment of LS. METHODS: We compiled retrospectively the clinical data on 8 cases of jLS that were treated with abatacept in our academic center. A systematic review protocol was developed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA-P) guidelines and has been registered with the international prospective register of systematic reviews (PROSPERO). Standardized searches of MEDLINE/PubMed and EMBASE were undertaken to identify studies reporting the use of abatacept in the treatment of LS. Heterogeneity in study design, interventions and reported outcomes necessitated a qualitative data synthesis. RESULTS: The use of abatacept was effective and safe in our cohort of jLS patients. Our standardized searches identified 30 articles, of which 3 deemed eligible for full data extraction. All 3 studies were small (total of 18 patients; mean 6 subjects per study), single center, open-label, uncontrolled and non-randomized. The Risk of Bias Assessment Tool for Non-randomized Studies (RoBANS) identified high risk-of bias for confounding variables and blinding of assessors in each of the 3 studies evaluated and in our pediatric case series. CONCLUSIONS: The evidence-base to support the use of abatacept in the treatment of LS is currently limited and clinical practice guidelines should take a measured approach to such recommended therapy. Nonetheless, as the empirical evidence on the clinical effectiveness of abatacept in the treatment of LS accumulates, a double-blind placebo-controlled randomized clinical trial is necessary to formally evaluate the observations documented by case-based reports.

The Regulatory B Cell Compartment Expands Transiently During Childhood and Is Contracted in Children With Autoimmunity.

OBJECTIVE: Regulatory B cells that inhibit immune responses through interleukin-10 (IL-10) secretion (B10 cells) have been characterized in adult subjects with autoimmune disease. The aim of this study was to characterize B10 cells in individuals across the entire age range of normal human development and changes in their frequency and numbers in children with autoimmunity. METHODS: The phenotype and numbers of B10 cells in blood were examined in healthy individuals and children with autoimmunity, using flow cytometry. B10 cell function was assessed by measuring the effect of B cell-derived IL-10 on interferon-γ (IFNγ) expression by CD4+ T cells. Serum cytokine levels were measured by enzyme-linked immunosorbent assay. RESULTS: The frequency of B10 cells transiently increased during childhood, when up to 30% of B cells were competent to produce IL-10, compared with the low frequencies in healthy newborns (3-4%) and adults (7-9%). The surface phenotype of B10 cells in children revealed age-dependent variability. B10 cells from children were distinct from proinflammatory cytokine-producing B cells and down-regulated IFNγ production by CD4+ T cells in vitro. Compared with age-matched healthy controls, children with autoimmunity had lower numbers and frequencies of B10 cells (decreased by 39% and 48%, respectively), higher IFNγ levels, and lower IL-21 levels in serum. IFNγ inhibited, whereas IL-21 promoted, B cell IL-10 competence in vitro. CONCLUSION: B10 cells, a functionally defined cell subset with a variable surface phenotype reflective of overall B cell development, transiently expand during childhood. B10 cell frequencies and numbers were decreased in children with autoimmunity, which may be explained in part by alterations in serum IFNγ and IL-21 that differentially regulate B10 cell development.

IL-10-producing regulatory B cells (B10 cells) in autoimmune disease.

B cell abnormalities contribute to the development and progress of autoimmune disease. Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited to the production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells have other functions potentially relevant to autoimmunity. Such functions include antigen presentation to and activation of T cells, expression of co-stimulatory molecules and cytokine production. Recently, the ability of B cells to negatively regulate cellular immune responses and inflammation has been described and the concept of regulatory B cells has emerged. A variety of cytokines produced by regulatory B cell subsets have been reported, with IL-10 being the most studied. In this review, this specific IL-10-producing subset of regulatory B cells has been labeled B10 cells to highlight that the regulatory function of these rare B cells is mediated by IL-10, and to distinguish them from other B cell subsets that regulate immune responses through different mechanisms. B10 cells are a functionally defined subset currently identified only by their competency to produce and secrete IL-10 following appropriate stimulation. Although B10 cells share surface markers with other previously defined B cell subsets, currently there is no cell surface or intracellular phenotypic marker or set of markers unique to B10 cells. The recent discovery of an effective way to expand B10 cells ex vivo opens new horizons in the potential therapeutic applications of this rare B cell subset. This review highlights the current knowledge on B10 cells and discusses their potential as novel therapeutic agents in autoimmunity.