14 research outputs found

    Results of <i>MVK</i>, <i>NLRP3</i> and <i>TNFRSF1A</i> gene dosage analysis by qPCR-HRM (prospective study) and qPCR (retrospective study).

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    <p>We screened by qPCR-HRM (i) exons 2–8 and 10–11 of the <i>MVK</i> gene in 10 controls, 1 DNA sample with a 12q23.2 deletion, and 49 patients, (ii) the exon 3 of the <i>NLRP3</i> gene in 9 controls, 10 patients, and the artificial positive p.T348M patient and (iii) exons 2–4 of the <i>TNFRSF1A</i> gene in 13 controls and 54 patients. We screened by qPCR (i) exon 9 of the <i>MVK</i> gene in 10 controls, 1 DNA sample with a 12q23.2q24.11 deletion, and 12 patients and (ii) exons 2–9 of the <i>NLRP3</i> gene in 9 controls and 38 patients.</p><p></p>The red bars indicate the range limits of the control ratios, between 0.75 and 1.28.<p></p

    Controls used in this work.

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    <p><sup>(1)</sup> heterozogote, <sup>(2)</sup> homozygote, <sup>(3)</sup> compound heterozygote.</p><p>*These polymorphisms were not detected by HRM.</p>†<p>Copy number variation.</p

    Primers used in this study.

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    1<p>All primers (except HRM-qPCR MVK/9 primer) showed a PCR efficiency range between 80 and 100% (IC+/−0.2%).</p>2<p>qPCR and HRM of <i>MVK</i> exon 9 could not be performed in the same run due to low PCR efficiency. We used therefore two different primer pairs.</p>3<p>p.T348M allele specific primer.</p>4<p>Forward primers were 6FAM labeled. Nine distinct PCR sets (Multiplex A to I) were designed, each yielding a pattern of 3 to 6 peaks including the 2 control fragments (DMD and GFAP).</p

    Flow chart for the screening of mutations in the genes <i>MVK</i>, <i>NLRP3</i> and <i>TNFRSF1A</i>.

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    <p>In our patient cohort, we performed a simultaneous search for punctual mutations and gene rearrangements using qPCR-HRM in the prospective group and qPCR alone in the retrospective group (as these patients had already been assessed for qualitative alterations), then SQF-PCR in both groups as a confirmatory quantitative approach.</p

    Results of <i>MVK</i>, <i>NLRP3</i> and <i>TNFRSF1A</i> gene dosage analysis by SQF-PCR.

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    <p>We screened by SQF-PCR (i) all coding exons of the <i>MVK</i> gene in 9 controls, 1 DNA sample with a 12q23.2 deletion, and 28 patients (ii) all coding exons of the <i>NLRP3</i> gene in 9 controls and 86 patients and (iii) all coding exons of the <i>TNFRSF1A</i> gene in 9 controls and 54 patients.</p><p></p>The red bars indicate the range limits of the control ratios, between 0.75 and 1.25.<p></p

    Evolution of the number of alveolar fibrocytes in 4 patients with IPF where bronchoalveolar lavage fluid was obtained at two or more occasions.

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    <p>Alveolar fibrocytes were no more detectable in the 2 patients with initial detectable alveolar fibrocytes. In the 2 patients without detectable alveolar fibrocytes at initial evaluation, fibrocytes remained undetectable in subsequent broncholveolar lavage fluid analysis.</p

    Characterization of bronchoalveolar lavage cells in culture.

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    <p>A) Morphology of adherent bronchoalveolar lavage (BAL) cells at amplification when a positive culture occurred. From left to right: after 5 days (scale bar  =  25 ”m), at 10 days (scale bar  =  250 ”m) and at 21 days (scale bar  =  250 ”m). The arrows indicate typical adherent mesenchymal cells with high (*, fibrocyte phenotype) or low ratio (#, fibroblast phenotype) of cell length to cell width. <b>B)</b> Confocal microscopy analysis of fibrocytes (spindle-shaped cells co-expressing CD45 and collagen) in BAL cell culture at day 21. From left to right: CD45, collagen 1, thiazole orange protein 3 (TO-PRO-3) and merge of the three fluorescence channels. Scale bars  =  15 ”m. <b>C)</b> Representative immunocytochemical stain prepared from alveolar fibroblasts at passage 1. From left to right: Ig Isotype, α-propyl-4-hydroxylase (α-4H), collagen 1, α-smooth muscle actin (a-SMA). Scale bars  =  100 ”m.</p
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