Mycoplasma bovis-induced inhibition of bovine peripheral blood mononuclear cell proliferation is ameliorated after blocking the immune-inhibitory programmed death 1 receptor

Mycoplasma bovis-induced immune suppression is a major obstacle faced by the host for controlling infections. M. bovis impairment of antigen-specific T-cell responses is achieved through inhibiting the proliferation of peripheral blood mononuclear cells (PBMCs). This impairment may contribute to the persistence of M. bovis infection in various sites, including lungs, and its systemic spread to various organs such as joints, with the underlying mechanisms remaining elusive. Here, we elucidated the role of the immune-inhibitory receptor programmed death 1 (PD-1) and its ligand (PD-L1) in M. bovis infection. Flow cytometry (FCM) analyses revealed an upregulation of PD-L1 expression on tracheal and lung epithelial cell lines after M. bovis infection. In addition, we found increased PD-L1 expression on purified lung lavage macrophages following M. bovis infection by FCM and determined its localization by immunofluorescence analysis comparing infected and control lung tissue sections. Moreover, M. bovis infection increased the expression of the PD-1 receptor on total PBMCs and in gated CD4+ and CD8+ T-cell subpopulations. We demonstrated that M. bovis infection induced a significant decrease in CD4+ PD-1INT and CD8+ PD-1INT subsets with intermediate PD-1 expression, which functioned as progenitor pools giving rise to CD4+ PD-1HIGH and CD8+ PD-1HIGH subsets with high PD-1 expression levels. We blocked PD-1 receptors on PBMCs using anti-PD-1 antibody at the beginning of infection, leading to a significant restoration of the proliferation of PBMCs. Taken together, our data indicate a significant involvement of the PD-1/PD-L1 inhibitory pathway during M. bovis infection and its associated immune exhaustion, culminating in impaired host immune responses. - 2018 American Society for Microbiology.This work was made possible by financial support from the Alberta Livestock and Meat Agency Ltd. and the Saskatchewan Agriculture Development Fund. We also thank Don Wilson and the VIDO Animal Care Unit for their invaluable help in obtaining blood and lung lavage samples.Scopu

Performance and comparability of laboratory methods for measuring ferritin concentrations in human serum or plasma: A systematic review and meta-analysis

Background Different laboratory methods are used to quantify ferritin concentrations as a marker of iron status. A systematic review was undertaken to assess the accuracy and comparability of the most used methods for ferritin detection. Methods and findings National and regional databases were searched for prospective, retrospective, sectional, longitudinal and case-control studies containing the characteristics and performance of at least one method for serum/plasma ferritin determinations in humans published to date. The analysis included the comparison between at least 2 methods detailing: sensitivity, precision, accuracy, predictive values, inter-methods adjustment, and use of international reference materials. Pooled method performance was analyzed for each method and across methods. Outcomes Search strategy identified 11893 records. After de-duplication and screening 252 studies were assessed, including 187 studies in the qualitative analysis and 148 in the meta-analysis. The most used methods included radiometric, nonradiometric and agglutination assays. The overall within-run imprecision for the most reported ferritin methods was 6.2±3.4% (CI 5.69–6.70%; n = 171), between-run imprecision 8.9±8.7% (CI 7.44–10.35%; n = 136), and recovery rate 95.6% (CI 91.5–99.7%; n = 94). The pooled regression coefficient was 0.985 among all methods analyzed, and 0.984 when comparing nonradiometric and radiometric methods, without statistical differences in ferritin concentration ranging from 2.3 to 1454 µµg/L. Conclusion The laboratory methods most used to determine ferritin concentrations have comparable accuracy and performance. Registered in PROSPERO CRD42016036222

Construction of non-polar mutants in Haemophilus influenzae using FLP recombinase technology

Background Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium that causes otitis media in children as well as other infections of the upper and lower respiratory tract in children and adults. We are employing genetic strategies to identify and characterize virulence determinants in NTHi. NTHi is naturally competent for transformation and thus construction of most mutants by common methodologies is relatively straightforward. However, new methodology was required in order to construct unmarked non-polar mutations in poorly expressed genes whose products are required for transformation. We have adapted the lambda red/FLP-recombinase-mediated strategy used in E. coli for use in NTHi. Results A cassette containing a spectinomycin resistance gene and an rpsL gene flanked by FRT sites was constructed. A PCR amplicon containing 50 base pairs of DNA homologous to the 5' and 3' ends of the gene to be disrupted and the cassette was generated, then recombineered into the target NTHi gene, cloned on a plasmid, using the lambda recombination proteins expressed in E. coli DY380. Thus, the gene of interest was replaced by the cassette. The construct was then transformed into a streptomycin resistant NTHi strain and mutants were selected on spectinomycin-containing growth media. A plasmid derived from pLS88 with a temperature sensitive replicon expressing the FLP recombinase gene under the control of the tet operator/repressor was constructed. This plasmid was electroporated into the NTHi mutant at the permissive temperature and FLP expression was induced using anhydrotetracycline. The recombinase recognizes the FRT sites and eliminates the antibiotic cassette by site-specific recombination, creating the unmarked non-polar mutation. The plasmid is cured by growth of cells at the restrictive temperature. Conclusion The products of the genes in the NTHi pilABCD operon are required for type IV pilus biogenesis and have a role in transformation. We demonstrated the utility of our methodology by the construction of a non-polar pilA mutation in NTHi strain 2019 and complementation of the mutation with a plasmid containing the pilA gene. Utilization of this approach allowed us to readily generate unmarked non-polar mutations in NTHi genes.This work was supported by NIH grants R01DC007464 to RSM, R01DC003915 to Lauren Bakaletz and a subcontract from N01AI30040 to Michael Apicella. We thank Michael Apicella for the gifts of NTHi strains 2019 and 2019 rpsL

Activation of the SMU.1882 Transcription by CovR in Streptococcus mutans

In Streptococcus mutans, the global response regulator CovR plays an important role in biofilm formation, stress-tolerance response, and caries production. We have previously shown that CovR acts as a transcriptional repressor by binding to the upstream promoter regions of its target genes. Here, we report that in vivo, CovR activates the transcription of SMU.1882, which encodes a small peptide containing a double-glycine motif. We also show that SMU.1882 is transcriptionally linked to comA that encodes a putative ABC transporter protein. Several genes from man gene clusters that encode mannose phosphotranferase system flank SMU.1882 -comA genes. Genomic comparison with other streptococci indicates that SMU.1882 is uniquely present in S. mutans, while the man operon is conserved among all streptococci, suggesting that a genetic rearrangement might have taken place at this locus. With the use of a transcriptional reporter system and semi-quantitative RT-PCR, we demonstrated the transcriptional regulation of SMU.1882 by CovR. In vitro gel shift and DNase I foot-printing analyses with purified CovR suggest that CovR binds to a large region surrounding the -10 region of the P1882. Using this information and comparing with other CovR regulated promoters, we have developed a putative consensus binding sequence for CovR. Although CovR binds to P1882, in vitro experiments using purified S. mutans RpoD, E. coli RNA polymerase, and CovR did not activate transcription from this promoter. Thus, we speculate that in vivo, CovR may interfere with the binding of a repressor or requires a cofactor

Search for Neutral Higgs Bosons in Events with Multiple Bottom Quarks at the Tevatron

The combination of searches performed by the CDF and D0 collaborations at the Fermilab Tevatron Collider for neutral Higgs bosons produced in association with b quarks is reported. The data, corresponding to 2.6 fb-1 of integrated luminosity at CDF and 5.2 fb-1 at D0, have been collected in final states containing three or more b jets. Upper limits are set on the cross section multiplied by the branching ratio varying between 44 pb and 0.7 pb in the Higgs boson mass range 90 to 300 GeV, assuming production of a narrow scalar boson. Significant enhancements to the production of Higgs bosons can be found in theories beyond the standard model, for example in supersymmetry. The results are interpreted as upper limits in the parameter space of the minimal supersymmetric standard model in a benchmark scenario favoring this decay mode.Comment: 10 pages, 2 figure

Search for the standard model Higgs boson in the H to ZZ to 2l 2nu channel in pp collisions at sqrt(s) = 7 TeV

A search for the standard model Higgs boson in the H to ZZ to 2l 2nu decay channel, where l = e or mu, in pp collisions at a center-of-mass energy of 7 TeV is presented. The data were collected at the LHC, with the CMS detector, and correspond to an integrated luminosity of 4.6 inverse femtobarns. No significant excess is observed above the background expectation, and upper limits are set on the Higgs boson production cross section. The presence of the standard model Higgs boson with a mass in the 270-440 GeV range is excluded at 95% confidence level.Comment: Submitted to JHE

Genes Important for Catalase Activity in Enterococcus faecalis

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly

Rational Design of a Plasmid Origin That Replicates Efficiently in Both Gram-Positive and Gram-Negative Bacteria

Background: Most plasmids replicate only within a particular genus or family

Search for anomalous t t-bar production in the highly-boosted all-hadronic final state

A search is presented for a massive particle, generically referred to as a Z', decaying into a t t-bar pair. The search focuses on Z' resonances that are sufficiently massive to produce highly Lorentz-boosted top quarks, which yield collimated decay products that are partially or fully merged into single jets. The analysis uses new methods to analyze jet substructure, providing suppression of the non-top multijet backgrounds. The analysis is based on a data sample of proton-proton collisions at a center-of-mass energy of 7 TeV, corresponding to an integrated luminosity of 5 inverse femtobarns. Upper limits in the range of 1 pb are set on the product of the production cross section and branching fraction for a topcolor Z' modeled for several widths, as well as for a Randall--Sundrum Kaluza--Klein gluon. In addition, the results constrain any enhancement in t t-bar production beyond expectations of the standard model for t t-bar invariant masses larger than 1 TeV.Comment: Submitted to the Journal of High Energy Physics; this version includes a minor typo correction that will be submitted as an erratu