Project description:Filamentous hemagglutinin (FHA), Bordetella pertussis' key adhesin, is both present at the cell surface and released in the extracellular milieu. To comprehend the role free FHA plays in the pathogenic process, we analyzed the global transcriptional changes in human peripheral blood mononuclear cells (PBMC) after exposure to that purified virulence factor. This analysis was undertaken with four different FHA preparations (FHA-1, FHA-2, FHA-3, and FHA-4), purified from four different B. pertussis strains. Cultures comprising 3x106 PBMC were stimulated for 0.5, 2, 4, and 6 hours with 5 ug/ml of each purified B. pertussis FHA. Control PBMC cultures were simultaneously treated with similar volumes of elution buffers (Mock-1 and Mock-2), and sampled at the same time points. The array experiment was composed of 27 samples. Most of the transcripts known to be part of the "common host-transcriptional response" that mediates inflammation were among the 1,235 array elements (representing 683 known unique genes) showing a greater than 3-fold change in transcript abundance between FHA-treated and untreated cells. We also identified an FHA-specific signature not observed in cells stimulated with B. pertussis LPS or heat-killed B. pertussis. This response, mounted by 13 genes, largely (69%, 9/13 genes) interferon (IFN)-regulated genes. Further analysis revealed that 18.3% (125 out of 683) of the FHA-responsive genes were in fact regulated by IFN. These included several major players of the antiviral IFN type I response, as well as three key components of the ISGylation pathway. Induction of the ubiquitin-like protein ISG15 and its specific protease USP18 was confirmed at the mRNA level by relative real-time polymerase chain reaction (RT-PCR). Western-blot analysis demonstrated the presence of both free ISG15 and several ISGylated conjugates in FHA-stimulated PBMC cell lysate. Intracellular FACS analysis provided further evidence that monocytes and a natural-killer (NK)-enriched cell population are the primary producers of ISG15 after FHA stimulation. Our results revealed new activities of free B. pertussis FHA: activation of the host IFN and ISGylation pathways. The consequences of such activation are yet to be resolved, but we hypothesize that FHA utilizes the ISGylation pathway or USP18 to regulate the IFN response, which in turn sensitizes the host to the infection and induces cell death. Groups of assays that are related as part of a time series. Infection: treatment with purified B. pertussis filamentous hemagglutinin (FHA) at 0.005 mg/mL Time: Samples were collected after various treatment time (hours) Keywords: time_series_design Computed

Project description:The aim of the study was to address the concept of field cancerization in oral cancer. The presence of genomic aberrations, indicative of chromosomal instability (CIN), in oral distant fields (ODFs) of visually normal and non-dysplastic mucosa at the mirror image from concomitant oral potentially malignant lesions (OPMLs) was investigated. This pilot study comprised 16 OPMLs (8 without dysplasia, nd-OPMLs; 8 with dysplasia, d-OPMLs) and 16 ODFs. DNA diploid (DNA Index, DI=1) and aneuploid (DIM-bM-^IM- 1) sublines were detected by high resolution DNA-flow cytometry (FCM) at (hr DNA-FCM) using DAPI stained nuclei suspensions. Nuclei with different DIs were FCM-sorted in order to enrich the epithelial component and to obtain genomic DNA for high resolution oligonucleotide array-Comparative Genomic Hybridization (a-CGH) analysis to provide a genome-wide measurement of DNA copy number aberrations (CNAs). The frequencies of DNA aneuploidy in ODFs and OPMLs were 6.2% and 43.8%, respectively (p=0.037). ODFs and nd-OPMLs were all near-diploid (DIM-bM-^IM- 1 and DIM-bM-^IM-$1.4), while d-OPMLs were also high-aneuploid (DI>1.4). CNA averages were 2.3 in ODFs (1.5 for nd-OPMLs and 3.1 for d-OPMLs), and 7.325 in OPMLs (3.0 in nd-OPMLs; 11.6 in d-OPMLs). CNAs were present in the DNA diploid sublines and often the same CNAs were observed in both ODFs and corresponding OPMLs DNA aneuploid sublines and CNAs in the present series of 16 ODFs are likely to represent early events of the natural history of oral carcinogenesis and to indicate an early onset of the field effect cancerization. Moreover, gains within 20q13.33-qter, 7p22.2-pter and 16p13.3-pter chromosomal regions in ODFs and in the relative OPMLs suggest that specific genes localized in these regions (RTEL1, MAD1L1 and TEL2) might contribute to the ODF/d-OPML transition. We analyzed: 8 samples of oral potentially malignant lesions with dysplasia, 8 samples of oral potentially malignant lesions without dysplasia and for each patient a corresponding oral distant field of visually normal mucosa.

Project description:Resurgence of pertussis has been observed in many countries with high vaccination coverage and clonal expansion of certain Bordetella pertussis strains has been associated with recent epidemics in Europe. It is known that vaccinations have selected strains which are different from those used for vaccine production. However, little is known about the differences in genomic content of strains circulating before the vaccination was introduced. In this study, we compared the genomes of 39 vaccine strains and old clinical isolates collected from Finland (N=5), Poland (N=14), Serbia (N=10) and the UK (N=10). The analysis included genotyping, pulsed-field gel electrophoresis (PFGE) and comparative genomic hybridisation (CGH). Compared to the strain Tohama I, the European strains analyzed have lost three major regions of difference (RD3, 5 and 29). However, difference in frequency of the absent RDs 3, 5 or 29 was observed among the four countries. Of the strains with absent RD5, half had also a duplicated region in the genome. All four RDs (RD22, 23, 24 and 26) absent in Tohama I were present in majority of the tested strains. Results obtained from PFGE analysis correlated well with those of CGH. Recently a novel pertussis toxin promoter allele (ptxP3) was described. Strains with ptxP3 have replaced resident ptxP1 strains. When the recent strains (N=22) selected from the four countries were examined, the ptxP3 allele was found in all countries except Poland. Our result indicates that at least three clusters of B. pertussis circulated in Europe in pre-vaccine era and their genomes were distinct from that of the reference strain Tohama I. Although progressive gene loss occurs in B. pertussis population with time, difference in frequency of the lost genes were observed among the countries. The observed differences in genomic content might be vaccine-driven. The strains were serotyped for the fimbriae, genotyped with LightCycler RT-PCR for pertussis toxin subunit 1 (ptxA), pertussis toxin promoter (ptxP) (Kallonen et al 2011, unpublished data, submitted) and pertactin (prn). The vaccine and old strains were assigned to profiles according to the PFGE results. From every PFGE profile from all countries at least 1 strain was selected and tested for CGH. Altogether 29 strains were analysed with CGH from Poland, Serbia and the United Kingdom, 12, 7 and 10, respectively. In addition 5 old previously published Finnish strains were included in the analysis as well as the reference strain Tohama I (Heikkinen et al., 2007). The hybridisations were performed as previously described (Heikkinen et al., 2007). Microarray data analysis was performed with R/Bioconductor (R Development Core Team, 2008). Print-tip-loess normalization was performed with default parameters and the gains/losses were filtered with Limma package. The regions of difference (RDs) found in this study are presented with the nomenclature defined by Brinig et al. (2006). The RDs which were not described by Brinig et al are indicated by RDs and the consequent unused number.

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Bordetella pertussis CGH

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