Biochemical properties of Paracoccus denitrificans FnrP:Reactions with molecular oxygen and nitric oxide

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe-4S] cluster containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~6-fold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer-dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe-4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers

An update on molecular cat allergens: Fel d 1 and what else? Chapter 1: Fel d 1, the major cat allergen

Background: Cats are the major source of indoor inhalant allergens after house dust mites. The global incidence of cat allergies is rising sharply, posing a major public health problem. Ten cat allergens have been identified. The major allergen responsible for symptoms is Fel d 1, a secretoglobin and not a lipocalin, making the cat a special case among mammals. Main body: Given its clinical predominance, it is essential to have a good knowledge of this allergenic fraction, including its basic structure, to understand the new exciting diagnostic and therapeutic applications currently in development. The recent arrival of the component-resolved diagnosis, which uses molecular allergens, represents a unique opportunity to improve our understanding of the disease. Recombinant Fel d 1 is now available for in vitro diagnosis by the anti-Fel d 1 specific IgE assay. The first part of the review will seek to describe the recent advances related to Fel d 1 in terms of positive diagnosis and assessment of disease severity. In daily practice, anti-Fel d 1 IgE tend to replace those directed against the overall extract but is this attitude justified? We will look at the most recent arguments to try to answer this question. In parallel, a second revolution is taking place thanks to molecular engineering, which has allowed the development of various forms of recombinant Fel d 1 and which seeks to modify the immunomodulatory properties of the molecule and thus the clinical history of the disease via various modalities of anti-Fel d 1-specific immunotherapy. We will endeavor to give a clear and practical overview of all these trends

Identification of an immunodominant region of the major house dust mite allergen Der p 2 presented by common human leucocyte antigen alleles.

BACKGROUND: Better understanding of the relevance of the immune response to common environmental allergens, such as the major house dust mite (HDM) allergen Der p 2, requires characterization of constituent T-cell epitopes. AIM: To identify CD4(+) T-cell epitopes within Der p 2 recognized by commonly expressed human leucocyte antigen (HLA) alleles. METHODS: HLA-blocking antibodies, peptide pools and truncations were used in ELISpot assays to establish restricted T-cell epitopes. RESULTS: People with and without atopic dermatitis have detectable Der p 2-specific T cells in the peripheral blood, which can proliferate in response to Der p 2 peptides. Interleukin-4-specific responses, both ex vivo and cultured to Der p 2 peptides, had a significant positive correlation with HDM-specific serum IgE. Within one pool of Der p 2 peptides, the 20mer D11 was found to induce multiple responses restricted through several alleles, including HLA-DPB1*0401 and HLA-DRB1*01. CONCLUSIONS: We have identified an immunogenic region of Der p 2 presented by common HLA class II alleles, including the most commonly expressed HLA allele DPB1*0401. Identification of such epitopes may be of future value in peptide immunotherapeutic approaches

Phenotypic analysis of perennial airborne allergen-specific CD4 + T cells in atopic and non-atopic individuals

Background Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4 + T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. Objective To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4 + T cells in adult individuals with severe persistent AD in comparison with healthy controls. Methods Using HLA class II tetrameric complexes based on a HLA-DPB1 *0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. Results Ex vivo Fel d 1-specific DPB1 *0401-restricted CD4 + T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. Conclusions and Clinical Relevance Circulating Fel d 1-specific DPB1 *0401-restricted CD4 + T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals. © 2011 Blackwell Publishing Ltd

Human antimicrobial peptides LL-37 and human β-defensin-2 reduce viral replication in keratinocytes infected with varicella zoster virus

Background. There is mounting evidence that antimicrobial peptides have an important role in cutaneous defence, but the expression of these antimicrobial peptides in atopic eczema (AE) is still unclear. There are several families of antimicrobial peptides, including cathelicidins and human β-defensins. Patients with AE are more susceptible to severe cutaneous viral infections, including varicella zoster virus (VZV). Aim. To characterize the functional activity of the antimicrobial peptides LL-37 (human cathelicidin) and human β-defensin (hBD)-2 keratinocytes were infected with VZV, in a skin-infection model. Methods. Flow-cytometry analysis was used to investigate LL-37 expression in normal human keratinocytes, and quantitative PCR was used to determine viral loads in infected HaCaT keratinocytes and B cells, with and without exogenous LL-37 and hBD-2. Results. LL-37 expression was present in keratinocytes, and both exogenous LL-37 and hBD-2 significantly reduced VZV load in infected keratinocytes and B cells. Specific antibodies blocked the antiviral action exhibited by these antimicrobial peptides. Preincubation of VZV with LL-37, but not hBD-2, further reduced VZV load. Conclusions. Both LL-37 and hBD-2 have an antiviral effect on VZV replication in the keratinocyte HaCaT cell line and in B cells, but their mechanism of action is different. Evidence of the relationship between antimicrobial peptide expression and higher susceptibility to infections in AE skin is still emerging. Developing novel antiviral therapies based on antimicrobial peptides may provide improved treatment options for patients with AE. © The Author(s) CED © 2012 British Association of Dermatologists

The National Lung Matrix Trial: translating the biology of stratification in advanced non-small-cell lung cancer.

Background The management of NSCLC has been transformed by stratified medicine. The National Lung Matrix Trial (NLMT) is a UK-wide study exploring the activity of rationally selected biomarker/targeted therapy combinations.Patients and methods The Cancer Research UK (CRUK) Stratified Medicine Programme 2 is undertaking the large volume national molecular pre-screening which integrates with the NLMT. At study initiation, there are eight drugs being used to target 18 molecular cohorts. The aim is to determine whether there is sufficient signal of activity in any drug-biomarker combination to warrant further investigation. A Bayesian adaptive design that gives a more realistic approach to decision making and flexibility to make conclusions without fixing the sample size was chosen. The screening platform is an adaptable 28-gene Nextera next-generation sequencing platform designed by Illumina, covering the range of molecular abnormalities being targeted. The adaptive design allows new biomarker-drug combination cohorts to be incorporated by substantial amendment. The pre-clinical justification for each biomarker-drug combination has been rigorously assessed creating molecular exclusion rules and a trumping strategy in patients harbouring concomitant actionable genetic abnormalities. Discrete routes of pathway activation or inactivation determined by cancer genome aberrations are treated as separate cohorts. Key translational analyses include the deep genomic analysis of pre- and post-treatment biopsies, the establishment of patient-derived xenograft models and longitudinal ctDNA collection, in order to define predictive biomarkers, mechanisms of resistance and early markers of response and relapse.Conclusion The SMP2 platform will provide large scale genetic screening to inform entry into the NLMT, a trial explicitly aimed at discovering novel actionable cohorts in NSCLC.Clinical trial isrctn 38344105