26 research outputs found
Immuno-localization of CD18 upon treatment with gentamicin.
<p>The two patients' Epstein-Barr virus-transformed cells were treated with gentamicin (1000 µg/ml) for 5 days. Cells were cytospun, fixed and immunostained with mouse monoclonal anti integrin β2 (CD18) antibody (Santa Cruz Biotechnology Inc., USA). Slides were mounted using immunofluorescence and DAPI for nuclear staining and analyzed by optic grid fluorescence microscopy (Olympus). <b>a.</b> 1000-fold magnification of healthy control's Epstein-Barr virus-transformed lymphocytes <b>b.</b> 600-fold magnification of gentamicin untreated (upper panel) and treated (lower panel) lymphoblastoids derived from patients 1 and 2. <b>c.</b> 1000-fold magnification of gentamicin untreated (left panel) and treated (right panel) lymphoblastoids derived from patient 1. N indicates nucleus; PM, plasma membrane; C, cytoplasm.</p
Theoretical model of human CD18.
<p><b>A.</b> A 3-D model of CD18 (blue) was built by homology modeling using ITGβ3 (CD61) structure as a template (red, PDB code 3ije). <b>B.</b> Superposition of the model with αvβ3 (CD51/CD61) complex (PDB code 1l5g). The β-I domains (CD18 in the model [blue]), CD61 in the template [red]) are given in cartoon representation with the arrow pointing to the position of the R188 residue, mutated in the LAD1 patients. The ITGαv subunit (CD51) is displayed in a surface representation.</p
<i>In vitro</i> induction of CD18 protein expression by gentamicin treatment.
<p>The two patients' Epstein-Barr virus-transformed cells were treated with increasing concentrations of gentamicin for 3 days. Proteins were extracted from whole cell lysates for western blot analysis (upper panels) using anti-CD18 antibodies (abcam) and anti-tubulin antibody (Sigma) for equal loading control. Intensities of the CD18 bands were calculated using Image EZ-Quant software package (EZ-Quante LTD., Israel), normalized to the untreated samples (lower panels).</p
CD18 and CD11b cell surface expression upon gentamicin treatment <i>in vivo</i>.
<p>Whole blood samples from patient #1 (a), patient #2 (b) and healthy control (c) were incubated with anti-CD18 or anti-CD11b antibodies (Coulter Diagnostics). Lymphocytes (Lymph.) and granulocytes (Granul.) were gated and the expression of CD18 or CD11b on their cell surface was measured using flow cytometry (Epics V; Coulter Electronics, Hialeah, FL or Becton Dickinson CANTO II, BD Biosciences, NJ, USA, Diva software). Unstained cells obtained from patient #1 (a) were used as the control to set the M1 threshold. The percent of cells expressing the relevant cell surface marker in each case is indicated.</p
R188X nonsense mutation in ITGβ2/CD18.
<p><b>a.</b> DNA sequences of the mutation region in exon 6 of CD18 gene in the two patients, their parents, one sister of patient #2 and one brother of patient #2. <b>b</b>. cDNA sequences of the mutation region in CD18 transcripts in the two patients <b>c.</b> Schematic structure of ITGβ2/CD18 protein, its domains and the position of the premature stop mutation. PSI indicates the plexin-semaphorin-integrin domain; HD, hybrid domain; EGFD, EGF-like domain; βTD, β-tail domain; PM, plasma membrane.</p
Expression of CD11/18 in patients 1 and 2 treated with or without gentamicin.
<p>Results are given in percents. When weak expression and/or low number of events were detected, MFI (mean fluorescence intensity) was measured as well, and results are presented in brackets.</p><p>Lymph indicates lymphocytes; Mono, monocytes; Gran, granulocytes.</p
Cumulative survival of AR patients compared to XL patients.
<p><i>*Log rank statistics for difference in survival between X-linked and AR: 7.01 (p = 0.0081)</i>.</p