Economic and health impact modelling of a whole genome sequencing-led intervention strategy for bacterial healthcare-associated infections for England and for the USA.

Bacterial healthcare-associated infections (HAIs) are a substantial source of global morbidity and mortality. The estimated cost associated with HAIs ranges from $35 to $45 billion in the USA alone. The costs and accessibility of whole genome sequencing (WGS) of bacteria and the lack of sufficiently accurate, high-resolution, scalable and accessible analysis for strain identification are being addressed. Thus, it is timely to determine the economic viability and impact of routine diagnostic bacterial genomics. The aim of this study was to model the economic impact of a WGS surveillance system that proactively detects and directs interventions for nosocomial infections and outbreaks compared to the current standard of care, without WGS. Using a synthesis of published models, inputs from national statistics, and peer-reviewed articles, the economic impacts of conducting a WGS-led surveillance system addressing the 11 most common nosocomial pathogen groups in England and the USA were modelled. This was followed by a series of sensitivity analyses. England was used to establish the baseline model because of the greater availability of underpinning data, and this was then modified using USA-specific parameters where available. The model for the NHS in England shows bacterial HAIs currently cost the NHS around £3 billion. WGS-based surveillance delivery is predicted to cost £61.1 million associated with the prevention of 74 408 HAIs and 1257 deaths. The net cost saving was £478.3 million, of which £65.8 million were from directly incurred savings (antibiotics, consumables, etc.) and £412.5 million from opportunity cost savings due to re-allocation of hospital beds and healthcare professionals. The USA model indicates that the bacterial HAI care baseline costs are around $18.3 billion. WGS surveillance costs $169.2 million, and resulted in a net saving of ca.$3.2 billion, while preventing 169 260 HAIs and 4862 deaths. From a 'return on investment' perspective, the model predicts a return to the hospitals of £7.83 per £1 invested in diagnostic WGS in the UK, and US$18.74 per $1 in the USA. Sensitivity analyses show that substantial savings are retained when inputs to the model are varied within a wide range of upper and lower limits. Modelling a proactive WGS system addressing HAI pathogens shows significant improvement in morbidity and mortality while simultaneously achieving substantial savings to healthcare facilities that more than offset the cost of implementing diagnostic genomics surveillance.

A Promyelocytic Leukemia Protein-Thrombospondin-2 Axis and the Risk of Relapse in Neuroblastoma.

PURPOSE: Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system with a complex biology, prone to metastasize and relapse. High-risk, metastatic cases are explained in part by amplification or mutation of oncogenes, such as MYCN and ALK, and loss of tumor suppressor genes in chromosome band 1p. However, it is fundamental to identify other pathways responsible for the large portion of neuroblastomas with no obvious molecular alterations. EXPERIMENTAL DESIGN: Neuroblastoma cell lines were used for the assessment of tumor growth in vivo and in vitro Protein expression in tissues and cells was assessed using immunofluorescence and IHC. The association of promyelocytic leukemia (PML) expression with neuroblastoma outcome and relapse was calculated using log-rank and Mann-Whitney tests, respectively. Gene expression was assessed using chip microarrays. RESULTS: PML is detected in the developing and adult sympathetic nervous system, whereas it is not expressed or is low in metastatic neuroblastoma tumors. Reduced PML expression in patients with low-risk cancers, that is, localized and negative for the MYCN proto-oncogene, is strongly associated with tumor recurrence. PML-I, but not PML-IV, isoform suppresses angiogenesis via upregulation of thrombospondin-2 (TSP2), a key inhibitor of angiogenesis. Finally, PML-I and TSP2 expression inversely correlates with tumor angiogenesis and recurrence in localized neuroblastomas. CONCLUSIONS: Our work reveals a novel PML-I-TSP2 axis for the regulation of angiogenesis and cancer relapse, which could be used to identify patients with low-risk, localized tumors that might benefit from chemotherapy. Clin Cancer Res; 22(13); 3398-409. ©2016 AACR.

Neisseria meningitidis Lacking the Major Porins PorA and PorB Is Viable and Modulates Apoptosis and the Oxidative Burst of Neutrophils.

The bacterial pathogen Neisseria meningitidis expresses two major outer-membrane porins. PorA expression is subject to phase-variation (high frequency, random, on-off switching), and both PorA and PorB are antigenically variable between strains. PorA expression is variable and not correlated with meningococcal colonisation or invasive disease, whereas all naturally-occurring strains express PorB suggesting strong selection for expression. We have generated N. meningitidis strains lacking expression of both major porins, demonstrating that they are dispensable for bacterial growth in vitro. The porAB mutant strain has an exponential growth rate similar to the parental strain, as do the single porA or porB mutants, but the porAB mutant strain does not reach the same cell density in stationary phase. Proteomic analysis suggests that the double mutant strain exhibits compensatory expression changes in proteins associated with cellular redox state, energy/nutrient metabolism, and membrane stability. On solid media, there is obvious growth impairment that is rescued by addition of blood or serum from mammalian species, particularly heme. These porin mutants are not impaired in their capacity to inhibit both staurosporine-induced apoptosis and a phorbol 12-myristate 13-acetate-induced oxidative burst in human neutrophils suggesting that the porins are not the only bacterial factors that can modulate these processes in host cells.

The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin.

UNLABELLED: Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid metabolism. In cells lacking RNase HI, replication initiates independently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI, origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics, and, ultimately, cell viability. IMPORTANCE: Cell division requires unwinding of millions of DNA base pairs to generate the template for RNA transcripts as well as chromosome replication. As both processes use the same template, frequent clashes are unavoidable. To minimize the impact of these clashes, transcription and replication in bacteria follow the same directionality, thereby avoiding head-on collisions. This codirectionality is maintained by a strict regulation of where replication is started. We have used Escherichia coli as a model to investigate cells in which the defined location of replication initiation is compromised. In cells lacking either RNase HI or RecG, replication initiates away from the defined replication origin, and we discuss the different mechanisms by which this synthesis arises. In addition, the resulting forks proceed in a direction opposite to normal, thereby inducing head-on collisions between transcription and replication, and we show that the resulting consequences are severe enough to threaten the viability of cells.

EXACT2: the semantics of biomedical protocols.

BACKGROUND: The reliability and reproducibility of experimental procedures is a cornerstone of scientific practice. There is a pressing technological need for the better representation of biomedical protocols to enable other agents (human or machine) to better reproduce results. A framework that ensures that all information required for the replication of experimental protocols is essential to achieve reproducibility. To construct EXACT2 we manually inspected hundreds of published and commercial biomedical protocols from several areas of biomedicine. After establishing a clear pattern for extracting the required information we utilized text-mining tools to translate the protocols into a machine amenable format. We have verified the utility of EXACT2 through the successful processing of previously 'unseen' (not used for the construction of EXACT2)protocols. METHODS: We have developed the ontology EXACT2 (EXperimental ACTions) that is designed to capture the full semantics of biomedical protocols required for their reproducibility. RESULTS: The paper reports on a fundamentally new version EXACT2 that supports the semantically-defined representation of biomedical protocols. The ability of EXACT2 to capture the semantics of biomedical procedures was verified through a text mining use case. In this EXACT2 is used as a reference model for text mining tools to identify terms pertinent to experimental actions, and their properties, in biomedical protocols expressed in natural language. An EXACT2-based framework for the translation of biomedical protocols to a machine amenable format is proposed. CONCLUSIONS: The EXACT2 ontology is sufficient to record, in a machine processable form, the essential information about biomedical protocols. EXACT2 defines explicit semantics of experimental actions, and can be used by various computer applications. It can serve as a reference model for for the translation of biomedical protocols in natural language into a semantically-defined format.

The human myometrium differentially expresses mTOR signalling components before and during pregnancy: evidence for regulation by progesterone.

Emerging studies implicate the signalling of the mammalian target of rapamycin (mTOR) in a number of reproductive functions. To this date, there are no data regarding the expression of mTOR signalling components in the human myometrium during pregnancy. We hypothesized that mTOR-related genes might be differentially expressed in term or preterm labour as well as in labour or non-labour myometria during pregnancy. Using quantitative RT-PCR we demonstrate for first time that there is a significant downregulation of mTOR, DEPTOR, and Raptor in preterm labouring myometria when compared to non-pregnant tissues taken from the same area (lower segment). We used an immortalized myometrial cell line (ULTR) as an in vitro model to dissect further mTOR signalling. In ULTR cells DEPTOR and Rictor had a cytoplasmic distribution, whereas mTOR and Raptor were detected in the cytoplasm and the nucleus, indicative of mTORC1 shuttling. Treatment with inflammatory cytokines caused only minor changes in gene expression of these components, whereas progesterone caused significant down-regulation. We performed a non-biased gene expression analysis of ULTR cells using Nimblegen human gene expression microarray (n=3), and selected genes were validated by quantitative RT-PCR in progesterone treated myometrial cells. Progesterone significantly down-regulated key components of the mTOR pathway. We conclude that the human myometrium differentially expresses mTOR signalling components and they can be regulated by progesterone. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

A role for BELLRINGER in cell wall development is supported by loss-of-function phenotypes.

BACKGROUND: Homeodomain transcription factors play critical roles in metazoan development. BELLRINGER (BLR), one such transcription factor, is involved in diverse developmental processes in Arabidopsis, acting in vascular differentiation, phyllotaxy, flower and fruit development. BLR also has a redundant role in meristem maintenance. Cell wall remodelling underpins many of these processes, and BLR has recently been shown to regulate expression of PECTIN METHYL-ESTERASE 5 (PME5), a cell wall modifying enzyme in control of phyllotaxy. We have further explored the role of BLR in plant development by analysing phenotypes and gene expression in a series of plants over-expressing BLR, and generating combinatorial mutants with blr, brevipedicellus (bp), a member of the KNOX1 family of transcription factors that has previously been shown to interact with blr, and the homeodomain transcription factor revoluta (rev), required for radial patterning of the stem. RESULTS: Plants over-expressing BLR exhibited a wide range of phenotypes. Some were defective in cell size and demonstrated misregulation of genes predominantly affecting cell wall development. Other lines with more extreme phenotypes failed to generate lateral organs, consistent with BLR repressing transcription in the shoot apex. Cell wall dynamics are also affected in blr mutant plants, and BLR has previously been shown to regulate vascular development in conjunction with BP. We found that when bp and blr were combined with rev, a set of defects was observed that were distinct from those of bp blr lines. In these triple mutants xylem development was most strikingly affected, resulting in an almost complete lack of vessels and xylem parenchyma with secondary thickening. CONCLUSIONS: Our data support a role for BLR in ordering the shoot apex and, in conjunction with BP and REV, playing a part in determining the composition and organisation of the vascular system. Microarray analysis strongly indicates that the striking vascular phenotypes of blr bp rev triple mutants and plants over-expressing BLR result from the misregulation of a suite of genes, targets of BLR in wild type plants, that determine cell size and structure in the developing vasculature.

Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis.

The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Šresolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.

Primary cutaneous anaplastic large cell lymphoma shows a distinct miRNA expression profile and reveals differences from tumor-stage mycosis fungoides.

The miRNA expression profiles of skin biopsies from 14 primary cutaneous anaplastic large cell lymphoma (C-ALCL) patients were analysed with miRNA microarrays using the same control group of 12 benign inflammatory dermatoses (BID) as previously used to study the miRNA expression profile of tumor-stage mycosis fungoides (MF). We identified 13 differentially expressed miRNAs between C-ALCL and BID. The up-regulation of miR-155, miR-27b, miR-30c and miR-29b in C-ALCL was validated by miRNA-Q-PCR on independent study groups. Additionally, the miRNA expression profiles of C-ALCL were compared with those of tumor-stage MF. Although miRNA microarray analysis did not identify statistically significant differentially expressed miRNAs, miRNA-Q-PCR demonstrated statistically significantly differential expression of miR-155, miR-27b, miR-93, miR-29b and miR-92a between tumor-stage MF and C-ALCL. This study, the first describing the miRNA expression profile of C-ALCL, reveals differences with tumor-stage MF, suggesting a different contribution to the pathogenesis of these lymphomas.

ERF5 and ERF6 play redundant roles as positive regulators of JA/Et-mediated defense against Botrytis cinerea in Arabidopsis.

The ethylene response factor (ERF) family in Arabidopsis thaliana comprises 122 members in 12 groups, yet the biological functions of the majority remain unknown. Of the group IX ERFs, the IXc subgroup has been studied the most, and includes ERF1, ERF14 and ORA59, which play roles in plant innate immunity. Here we investigate the biological functions of two members of the less studied IXb subgroup: ERF5 and ERF6. In order to identify potential targets of these transcription factors, microarray analyses were performed on plants constitutively expressing either ERF5 or ERF6. Expression of defense genes, JA/Et-responsive genes and genes containing the GCC box promoter motif were significantly upregulated in both ERF5 and ERF6 transgenic plants, suggesting that ERF5 and ERF6 may act as positive regulators of JA-mediated defense and potentially overlap in their function. Since defense against necrotrophic pathogens is generally mediated through JA/Et-signalling, resistance against the fungal necrotroph Botrytis cinerea was examined. Constitutive expression of ERF5 or ERF6 resulted in significantly increased resistance. Although no significant difference in susceptibility to B. cinerea was observed in either erf5 or erf6 mutants, the erf5 erf6 double mutant showed a significant increase in susceptibility, which was likely due to compromised JA-mediated gene expression, since JA-induced gene expression was reduced in the double mutant. Taken together these data suggest that ERF5 and ERF6 play positive but redundant roles in defense against B. cinerea. Since mutual antagonism between JA/Et and salicylic acid (SA) signalling is well known, the UV-C inducibility of an SA-inducible gene, PR-1, was examined. Reduced inducibilty in both ERF5 and ERF6 constitutive overexepressors was consistent with suppression of SA-mediated signalling, as was an increased susceptibility to avirulent Pseudomonas syringae. These data suggest that ERF5 and ERF6 may also play a role in the antagonistic crosstalk between the JA/Et and SA signalling pathways.

The use of the pan-Neisseria microarray and experimental design for transcriptomics studies of Neisseria.

The pan-Neisseria microarray was the first bacterial microarray to address multiple strains and species, and is a tool specifically developed for the performance of comparative studies within and between species. To achieve this, its design was based upon a detailed comparison of multiple genomes, prior to probe selection, and serial triage to optimize sensitivity and specificity. While this tool can be used for transcriptional comparisons of the same species, such as isogenic mutants, or strains exposed to different environmental conditions, its features are also particularly suited to population and functional studies of unrelated strains. The optimal use of these tools, including the use of single-channel labeling for genomic studies, the biological replication needed to perform robust transcription studies, and key aspects of data analysis such as the use of cross-channel correction and Bayesian analytical approaches, is discussed.

Transcriptomic analysis reveals calcium regulation of specific promoter motifs in Arabidopsis.

Increases in intracellular calcium concentration ([Ca(2+)](c)) mediate plant responses to stress by regulating the expression of genes encoding proteins that confer tolerance. Several plant stress genes have previously been shown to be calcium-regulated, and in one case, a specific promoter motif Abscisic Acid Responsive-Element (ABRE) has been found to be regulated by calcium. A comprehensive survey of the Arabidopsis thaliana transcriptome for calcium-regulated promoter motifs was performed by measuring the expression of genes in Arabidopsis seedlings responding to three calcium elevations of different characteristics, using full genome microarray analysis. This work revealed a total of 269 genes upregulated by [Ca(2+)](c) in Arabidopsis. Bioinformatic analysis strongly indicated that at least four promoter motifs were [Ca(2+)](c)-regulated in planta. We confirmed this finding by expressing in plants chimeric gene constructs controlled exclusively by these cis-elements and by testing the necessity and sufficiency of calcium for their expression. Our data reveal that the C-Repeat/Drought-Responsive Element, Site II, and CAM box (along with the previously identified ABRE) promoter motifs are calcium-regulated. The identification of these promoter elements targeted by the second messenger intracellular calcium has implications for plant signaling in response to a variety of stimuli, including cold, drought, and biotic stress.

MicroRNA expression in multiple myeloma is associated with genetic subtype, isotype and survival.

BACKGROUND: MicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. However, the role of microRNAs in the pathogenesis of multiple myeloma (MM) is only poorly understood. We therefore used microarray analysis to elucidate the complete miRNome (miRBase version 13.0) of purified tumor (CD138+) cells from 33 patients with MM, 5 patients with monoclonal gammopathy of undetermined significance (MGUS) and 9 controls. RESULTS: Unsupervised cluster analysis revealed that MM and MGUS samples have a distinct microRNA expression profile from control CD138+ cells. The majority of microRNAs aberrantly expressed in MM (109/129) were up-regulated. A comparison of these microRNAs with those aberrantly expressed in other B-cell and T-cell malignancies revealed a surprising degree of similarity (~40%) suggesting the existence of a common lymphoma microRNA signature. We identified 39 microRNAs associated with the pre-malignant condition MGUS. Twenty-three (59%) of these were also aberrantly expressed in MM suggesting common microRNA expression events in MM progression. MM is characterized by multiple chromosomal abnormalities of varying prognostic significance. We identified specific microRNA signatures associated with the most common IgH translocations (t(4;14) and t(11;14)) and del(13q). Expression levels of these microRNAs were distinct between the genetic subtypes (by cluster analysis) and correctly predicted these abnormalities in > 85% of cases using the support vector machine algorithm. Additionally, we identified microRNAs associated with light chain only myeloma, as well as IgG and IgA-type MM. Finally, we identified 32 microRNAs associated with event-free survival (EFS) in MM, ten of which were significant by univariate (logrank) survival analysis. CONCLUSIONS: In summary, this work has identified aberrantly expressed microRNAs associated with the diagnosis, pathogenesis and prognosis of MM, data which will prove an invaluable resource for understanding the role of microRNAs in this devastating disease.

Genome sequence of Rhodobacter sphaeroides Strain WS8N.

Rhodobacter sphaeroides is a metabolically diverse photosynthetic alphaproteobacterium found ubiquitously in soil and freshwater habitats. Here we present the annotated genome sequence of R. sphaeroides WS8N.

miRNA expression profiling of mycosis fungoides.

MicroRNAs (miRNAs) are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many malignancies including lymphoma. However, the role of miRNAs in the pathogenesis of T-cell lymphoid malignancies is poorly understood. Previously we examined the miRNA profile of Sézary syndrome (Sz), a leukemia of skin-homing memory T cells. In this study we determined the complete miRNome of mycosis fungoides (MF), the most common type of cutaneous T cell lymphoma. The miRNA profile of skin biopsies from 19 patients with tumor stage MF and 12 patients with benign inflammatory dermatoses (eczema and lichen planus) were compared by microarray analysis. We identified 49 miRNAs that are differentially expressed in tumor stage MF compared to benign inflammatory dermatoses using ANOVA analysis (P < 0.05, Benjamini-Hochberg corrected). The majority of the differentially expressed miRNAs (30/49) were up-regulated in tumor stage MF. The most significant differentially expressed were miR-155 and miR-92a (both up-regulated in tumor stage MF), while miR-93 showed the highest up-regulation in tumor stage MF with a fold difference of 5.8. Differential expression of a selection of these miRNAs was validated by miRNA-Q-PCR on additional test groups (tumors and controls). None of the miRNAs up-regulated in tumor stage MF was previously shown to be up-regulated in Sz, and only 2 of the 19 miRNAs down-regulated in tumor stage MF were also down-regulated in Sz. Taken together this report is the first describing the miRNA signature of tumor stage MF.

Deep resequencing of serial sputum isolates of Mycobacterium tuberculosis during therapeutic failure due to poor compliance reveals stepwise mutation of key resistance genes on an otherwise stable genetic background.

OBJECTIVES: It has generally been held that the repeated emergence of resistance in Mycobacterium tuberculosis is due to the effects of large population sizes, slow replication, and prolonged colonization and treatment. However, there have been suggestions that its emergence is facilitated by high mutation rates due to a lack of mismatch repair, error-prone polymerases, and a potentially mutagenic host niche. Genome re-sequencing has indicated higher variability in strains with emergent resistance, but these studies have not been performed in serial isolates in which drug resistance has emerged. We have used genome re-sequencing to address the mutational processes that occur during the evolution of drug resistance during a clinical infection. METHODS: Serial isolates from a patient obtained over a 12 month period, and spanning the transition of the colonizing population from fully drug sensitive, to isoniazid resistant, to isoniazid and rifampicin (multiply drug) resistant, spanning an estimated minimum of 100 generations within the host, were deep sequenced using Illumina sequencing. The genomes were compared, and all mutations in non-repetitive sequences were identified. RESULTS: Specific mutations conferring resistance were identified. No additional mutations in non-repetitive regions were present. The mutations observed were kat S315T and rpoB D516Y. CONCLUSIONS: M. tuberculosis is relatively stable genetically within the host, and demonstrates greater stability than is suggested by in vitro studies of emergent drug resistance, or by models of hypermutability. This indicates that it is primarily the nature and duration of the infection that are sufficient to lead to the repeated emergence of drug resistance in this infection if improperly managed, and that the selective pressure of the drugs limits additional diversification. This emphasizes the central importance of maintaining therapeutic concentrations of at least two effective antibiotics for the duration of treatment to prevent the emergence of resistance.

MicroRNA expression in Sezary syndrome: identification, function, and diagnostic potential.

MicroRNAs are commonly aberrantly expressed in many cancers. Very little is known of their role in T-cell lymphoma, however. We therefore elucidated the complete miRNome of purified T cells from 21 patients diagnosed with Sézary Syndrome (SzS), a rare aggressive primary cutaneous T-cell (CD4(+)) lymphoma. Unsupervised cluster analysis of microarray data revealed that the microRNA expression profile was distinct from CD4(+) T-cell controls and B-cell lymphomas. The majority (104 of 114) of SzS-associated microRNAs (P < .05) were down-regulated and their expression pattern was largely consistent with previously reported genomic copy number abnormalities and were found to be highly enriched (P < .001) for aberrantly expressed target genes. Levels of miR-223 distinguished SzS samples (n = 32) from healthy controls (n = 19) and patients with mycosis fungoides (n = 11) in more than 90% of samples. Furthermore, we demonstrate that the down-regulation of intronically encoded miR-342 plays a role in the pathogenesis of SzS by inhibiting apoptosis, and describe a novel mechanism of regulation for this microRNA via binding of miR-199a* to its host gene. We also provide the first in vivo evidence for down-regulation of the miR-17-92 cluster in malignancy and demonstrate that ectopic miR-17-5p expression increases apoptosis and decreases cell proliferation in SzS cells.

The structure of a reduced form of OxyR from Neisseria meningitidis.

BACKGROUND: Survival of the human pathogen, Neisseria meningitidis, requires an effective response to oxidative stress resulting from the release of hydrogen peroxide by cells of the human immune system. In N. meningitidis, expression of catalase, which is responsible for detoxifying hydrogen peroxide, is controlled by OxyR, a redox responsive LysR-type regulator. OxyR responds directly to intracellular hydrogen peroxide through the reversible formation of a disulphide bond between C199 and C208 in the regulatory domain of the protein. RESULTS: We report the first crystal structure of the regulatory domain of an OxyR protein (NMB0173 from N. meningitidis) in the reduced state i.e. with cysteines at positions 199 and 208. The protein was crystallized under reducing conditions and the structure determined to a resolution of 2.4 A. The overall fold of the Neisseria OxyR shows a high degree of similarity to the structure of a C199S mutant OxyR from E. coli, which cannot form the redox sensitive disulphide. In the neisserial structure, C199 is located at the start of helix alpha3, separated by 18 A from C208, which is positioned between helices alpha3 and alpha4. In common with other LysR-type regulators, full length OxyR proteins are known to assemble into tetramers. Modelling of the full length neisserial OxyR as a tetramer indicated that C199 and C208 are located close to the dimer-dimer interface in the assembled tetramer. The formation of the C199-C208 disulphide may thus affect the quaternary structure of the protein. CONCLUSION: Given the high level of structural similarity between OxyR from N. meningitidis and E. coli, we conclude that the redox response mechanism is likely to be similar in both species, involving the reversible formation of a disulphide between C199-C208. Modelling suggests that disulphide formation would directly affect the interface between regulatory domains in an OxyR tetramer which in turn may lead to an alteration in the spacing/orientation of the DNA-binding domains and hence the interaction of OxyR with its DNA binding sites.

Tolerogenicity is not an absolute property of a dendritic cell.

Pharmacological modulation is known to temper the immune capacity of DC, enhancing the notion that modulated Ag-bearing DC might be used therapeutically to induce tolerance. We have investigated phenotypic features shared by such DC, and queried their potential to tolerize in different settings. Immature, IL-10, TGF-beta and 1alpha,25-dihydroxyvitamin D(3)-modulated BMDC all induced tolerance to male skin in female TCR transgenic A1.RAG mice, and the modulated DC also tolerized after exposure to the TLR4-ligand LPS. Transcript profiling revealed that this was achieved despite retaining much of the normal LPS-maturation response. No shared tolerance-associated transcripts could be identified. Equivalent BMDC could not tolerize in Marilyn TCR-transgenic mice. Simultaneous presentation of both A1.RAG and Marilyn peptide-Ag (Dby-H2E(k) and Dby-H2A(b)) on immature (C57BL/6JxCBA/Ca) F1 BMDC also only achieved tolerance in A1.RAG mice. Both strains registered Ag, but Foxp3(+) Treg were only induced in A1.RAG mice. In contrast, Marilyn T cells showed greater proliferation and an inflammatory bias, in response to Ag presented by immature F1 BMDC in vitro. In summary, while pharmacological agents can skew DC to reinforce their immature tolerogenic phenotype, the outcome of presentation is ultimately an integrated response including T-cell-intrinsic components that can over-ride for immune activation.