Parkin knockdown effect on NF-κB and MAPK signaling.

<p>(<b>A</b>) siRNA-transfected THP-1 macrophages were stimulated with 100 ng/ml LPS for the indicated times, then lysed and analysed by 12% SDS-PAGE followed by Western blotting. Membranes were stained for phospho-IκB-α then reprobed for IκB-ζ and β-actin (<b>B</b>) Nuclear extracts were also analyzed by transcription factor ELISA. THP-1 cells were treated with 100 ng/ml LPS for 4 hours, then extracted and analysed for NF-κB (p65) and AP-1 (phospho-c-Jun) binding to consensus DNA oligomers. Values are expressed as % binding of positive standard extracts and are representative of three experiments. Extracts from diluent (RPMI)-treated cells did not show DNA binding above background. (<b>C</b>) Membranes were stained for phospho-ERK1/2, phospho-p38 and phospho-JNK then reprobed for total JNK and β-actin. The JNK2 (p54) isoform is predominantly detected in these lysates although some phospho-JNK1 (p46) bands can be observed. Blots are representative of at least three experiments. (<b>D</b>) Transfected cells were pretreated with 30 µM JNK inhibitor SP600125 (SP) for 1 hour and then treated with LPS or diluent for 6 hours. IL-6 from the supernatants was measured by ELISA.</p

Whisker plots of <i>CCL2</i> and <i>IL6</i> transcript levels in whole blood cultures in the presence an absence of <i>M. leprae</i> sonicate.

<p>Whole blood from 62 Vietnamese subjects was stimulated with 10 µg/ml <i>M. leprae</i> sonicate and transcript levels of <i>CCL2</i> and <i>IL-6</i> were determined by real time PCR. (<b>A</b>) Transcript levels were normalized with the <i>HPRT</i> house keeping gene and expressed as ΔC_t in the absence (NON-STIM) and presence (STIM) of <i>M. leprae</i> sonicate. The median of the distribution is indicated by a solid line within the box. The resulting subdivision of the box indicates the distribution of the flanking 25% percentile in each direction while the error bars give the distribution of the upper and lower 25% of the ΔC_t values. (<b>B</b>) The increase of <i>CCL2</i> and <i>IL6</i> transcripts resulting from stimulation with <i>M. leprae</i> sonicate expressed as ΔΔC_t. Plots as described in A.</p

Parkin-silenced THP-1 macrophages cytokine screen.

<p>(<b>A</b>) Parkin was detected by indirect immunofluorescence of THP-1 cells following transfection with either scrambled siRNA (upper panel) or siRNA targeting Parkin (lower panel). Insets represent DAPI-stained nuclei. (<b>B</b>) PMA-differentiated THP-1 macrophages were transfected with control or Parkin-silencing siRNA. After 48 hours, cells were treated with H37Ra at an MOI of 10, <i>M. leprae</i> (ML) at an MOI 50, or left untreated (Neg.). After 6 hours, supernatants were collected and analyzed with a Milliplex 42-cytokine assay. Cytokines with detectable values (12 out of 42) are plotted on the graph. Cytokine production is expressed as ratio of cytokine secreted by cells transfected with siRNA for <i>PARK2</i> (Parkin) to cytokine secreted by cells transfected with control siRNA (scrambled). (<b>C</b>) PMA- differentiated THP-1 macrophages were transfected with control or Parkin-silencing siRNA. After 48 hours, cells were treated with LPS (10 ng/ml), <i>M. bovis</i> BCG at an MOI of 10, <i>M. leprae</i> (ML) at an MOI 50, or left untreated (Neg). After 6 hours supernatants were collected and analyzed with a Q-Plex custom cytokine multiplex assay. Values represent the ratio of concentrations produced by Parkin-silenced cells over controls ± SD of at least three independent experiments. (<b>D</b>) As described for <b>C</b> except that supernatants were collected after 24 hrs incubation with stimulants. * <i>p</i><0.05, non-parametric t test of unpaired samples.</p

Analysis of correlation between SNP genotypes and transcript expression in un-stimulated <i>ex vivo</i> whole blood cultures (ΔCt) or after <i>M. leprae</i> sonicate stimulation (ΔΔCt).

*<p>LD: Linkage disequilibrium; singleton SNPs (sSNP) and correlated SNPs (BIN 1, BIN 2 and BIN 3) are indicated.</p>**<p>Regression coefficients under dominant major allele model.</p>***<p><i>P</i>-values for significance of correlation between genotypes and transcript levels under a dominant major allele model employing a likelihood ratio test (LRT); significant correlations are in bold and underlined.</p

Parkin knockdown inhibits IL-6 and MCP-1 induction in macrophages and Schwann cells.

<p>PMA-differentiated THP-1 macrophages, VD-treated THP-1 macrophages, VD-treated human monocyte-derived macrophages and human Schwann cells were transfected with control or Parkin-silencing siRNA. Due to the limited number of cells available for each experiment, only one <i>PARK2</i> siRNA was used for MDM. After 48 hours, cells were treated with LPS (10 ng/ml), <i>M. bovis</i> BCG at an MOI of 10, <i>M. leprae</i> (ML) at an MOI 50, or left untreated (Neg.). Supernatants were collected after 6 hours and analyzed for (<b>A</b>) IL-6 and (<b>B</b>) MCP-1. The charts show the average concentration of cytokine in the supernatant in pg/ml ± SD of at least three independent experiments for each cell type. Scrambled: control siRNA, Parkin A: siRNA(A) for Parkin, Parkin B: siRNA(B) for Parkin. * <i>p</i><0.05, ** <i>p</i><0.01, non-parametric t test of unpaired samples.</p

Table indicating the clinical presentations of TB recorded in the fifty patients.

<p>The two patients presenting with IL-12Rβ1 deficiency are in bold. N means number and age is indicated in years.</p

Mendelian mutations in <i>IL12RB1</i> leading to severe tuberculosis in two kindreds.

<p><b>A</b>. Pedigree of the two families (A and B) with IL-12Rβ1 deficiency. Each generation is designated by a roman numeral (I–II), and each individual by an Arabic numeral. The double lines connecting the parents indicate consanguinity. The probands are indicated by an arrow, with black indicating <i>Mycobacterium tuberculosis</i> disease status. Individuals whose genetic status could not be evaluated are indicated by the symbol “E?”. <b>B</b>. Electrophoregram showing the genomic sequences of exons 9 and 5 in patients 1 and 2, respectively, compared with a control sequence. <b>C</b>. Schematic diagram of the coding region of the IL-12Rβ1 chain containing 17 coding exons and encoding a 662-amino acid protein with a leader sequence (exon1, L), extracellular domain (exons 2 to 13, EC), transmembrane domain (exon 14, TM) and an intracellular cytoplasmic domain (exons 15 to 17, IC). Published and unpublished mutations are indicated as follows: missense mutations are shown in purple, nonsense mutations are shown in red and complex mutations are shown in brown. Splicing mutations are shown in blue, large deletions are shown in green, insertions are shown in orange, and duplication is shown in magenta. * The 700+362_1619-944del mutation is the only mutation resulting in at the expression of a protein at the cell surface. Mutations of P1 (K305X) and P2 (R173W) are underlined. <b>D</b>. Chest X ray of patient 1 showing the localization of the disease.</p