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      Identification of a severe bleeding disorder in humans caused by a mutation in CalDAG-GEFI

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      The Journal of Experimental Medicine
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          Abstract

          Insight from William Muller In this issue, Canault et al. report for the first time a point mutation in the RAS guanyl-releasing protein 2 (RASGRP2) gene that results in a severe bleeding defect in humans. The study of inherited platelet disorders has shed light on the molecular mechanisms of physiologic thrombosis and hemostasis and led to the development of several therapies to prevent pathologic clotting. The RASGRP2 gene encodes the guanine nucleotide exchange factor (GEF) calcium- and DAG-regulated GEF 1 (CalDAG-GEFI), which is crucial for activation of Rap1, a small GTPase that regulates integrin-mediated activation and granule secretion in platelets and other cells. The function of CalDAG-GEFI has been studied in vitro and in mice—mice lacking CalDAG-GEFI have impaired thrombi formation by platelets as well as a defect in neutrophil function—but to date, no pathological RASGRP2 mutations have been identified in humans. Adhesion of calcein-AM–labeled platelets to fibrillar collagen under flow (750 s-1) from a homozygous (HOM), heterozygous (HET), and healthy (Ctl) subject. Percentage of covered area was assessed over 300 seconds (left). The initial 60 seconds are magnified (right). Now, Canault et al. have investigated the cause of an inherited platelet disorder in three siblings from a consanguineous marriage that are all affected by a severe bleeding disorder. Whole genome sequencing was used to identify a mutation (cG742T) in the RASGRP2 gene. This mutation reduces Rac1 GTP binding (secondary to decreased Rap1 activation), impairing the ability of platelets to aggregate in response to a variety of stimuli, to form thrombi under flow, and to undergo normal spreading. Heterozygotes also have platelets that fail to adhere normally under flow and have a spreading defect (see figure) but they do not suffer from bleeding because their platelets aggregate normally. The functional deficiency induced by the mutation was confined to platelets and megakaryocytes with no obvious alteration in leukocytes. This is probably because other GEFs in leukocytes are able to activate Rap1 or, as the authors speculate, that CalDAG-GEFI with this point mutation is still able to function in leukocytes. In fact, even in platelets, the aggregation defects can be overcome by high doses of agonists in vitro, so while CalDAG-GEFI may be the preferred way for platelets to activate Rap1, it is clearly not the only way. Mutations in CalDAG-GEFI had previously been reported to be responsible for leukocyte adhesion deficiency type III, which was later found to be caused by the absence of kindlin-3. To demonstrate that the mutation in CalDAG-GEFI is truly responsible for the phenotype, the authors showed that cells transfected with wild-type RASGRP2 can activate Rap1, whereas those transfected with the mutation found in the affected siblings cannot. Looking at these data from a different perspective, the presence of a single normal allele is sufficient to prevent bleeding, but not platelet adhesion to collagen. This suggests that partial inhibition of CalDAG-GEFI might be a novel and potentially safe therapeutic target to prevent thrombosis without causing bleeding—a holy grail of vascular medicine and surgery.

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          Human Cd25+Cd4+ T Regulatory Cells Suppress Naive and Memory T Cell Proliferation and Can Be Expanded in Vitro without Loss of Function

          Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4+ Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25+CD4+ Tr cells in humans has not been reported. Here we show that human CD25+CD4+ Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25+CD4+ Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25+CD4+ Tr cells strongly inhibited the proliferative responses of both naive and memory CD4+ T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25+CD4+ Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25+CD4+ Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.
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            Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding

            Inherited platelet disorders are rare diseases that give rise to bleeding when platelets fail to fulfill their hemostatic function upon vessel injury. Clinical manifestations include mainly mucocutaneous bleeding, menometrorrhagia and excessive bleeding after surgical intervention or trauma. The study of these diseases allows a better understanding of normal platelet biochemistry and physiology. These inherited disorders include abnormalities of platelet receptors, granules, or signal transduction (Nurden and Nurden, 2011). Signal transduction dysfunction is thought to be the most common cause of platelet inherited disorders; however, only a few have been successfully genotyped. Here, we have identified and characterized the first mutation of RASGRP2 (RAS guanyl-releasing protein-2) in a family suffering severe bleeding. RASGRP2 codes for a major signaling molecule in platelets, calcium-and-DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). It is a guanine nucleotide exchange factor (GEF) that is critical for Ras-like GTPase activation whose target is mainly Rap1 in platelets (Crittenden et al., 2004; Bergmeier et al., 2007; Cifuni et al., 2008; Stefanini et al., 2009). Rap1 is one of the most predominant small GTPases in platelets and constitutes a key signaling element that governs platelet activation by directly regulating integrin-mediated aggregation and granule secretion (Chrzanowska-Wodnicka et al., 2005; Zhang et al., 2011). Mice lacking CalDAG-GEFI not only have major defects in platelet function, with a reduced ability to form thrombi upon vascular injury, but they also have impaired neutrophil functions (Crittenden et al., 2004; Bergmeier et al., 2007; Carbo et al., 2010). To date, no pathological mutation in RASGRP2 has been reported in man and knowledge about the phenotype linked to human CalDAG-GEFI deficiency is lacking. RESULTS Initial characterization of the patients We report here the cases of three siblings (two males and one female) from consanguineous parents (first cousins) that were born between 1957 and 1962 after asymptomatic pregnancies. All suffered from mucocutaneous bleeding starting at 18 mo; these episodes consisted mainly of prolonged and severe epistaxis, hematomas, and bleeding after tooth extraction. The clinical presentation of the female was more severe due to massive menorrhagia and chronic anemia that were controlled by oestroprogestogenic therapy. Two of them required recurrent blood transfusions until adolescence. One of the patients successfully received transfusion of platelet concentrates before soft tissue surgery to prevent bleeding. Bleeding times tested on several occasions were always prolonged (>20 min). Interview and clinical examination of the parents, two other siblings, and the two children of one of the patient did not reveal any bleeding tendency. The bleeding times measured in the two unaffected siblings were normal (5–6 min). Initial platelet screening in patients demonstrated a reduced maximal aggregation in response to all tested doses of ADP or epinephrine and to low doses of thrombin receptor–activating peptide-14 (TRAP-14) and collagen, whereas aggregation with ristocetin or phorbol myristate acetate (PMA) was normal (Fig. 1 a). When induced by high-dose arachidonic acid, TRAP-14, or collagen, maximal aggregation was comparable to controls (Fig. 1 a). Notably, aggregation velocities were lower for the patient’s platelets as compared with healthy control platelets with all tested ADP doses and even at high doses of TRAP-14 and collagen (Fig. 1 b). Expression of all studied membrane receptors, including αIIbβ3 and GPIbα, were normal (Fig. 1 c). Binding of the αIIbβ3 activation-specific antibody, PAC-1, was identical for patients and controls at high (Fig. 1 c) and low (not depicted) doses of TRAP-14 and ADP. Platelet granule content and release were assayed. Platelet serotonin and PAI-1 contents, mepacrine uptake/release, and granulophysin (CD63) surface expression were normal upon high-dose agonist stimulation (ADP 10 µM and TRAP-14 50 µM; unpublished data). P-selectin surface expression tended to be lower in patients at high- (Fig. 1 c) and low-dose (not depicted) agonists. These results indicate normal contents of dense, α-, and lysosomal granules, albeit with a slightly reduced α-granule secretion. Clot retraction, induced by 1 U/ml thrombin was not affected (unpublished data). Thrombin generation in platelet-rich plasma (PRP) depends on platelet activation and αIIbβ3 activation as much as it does on the plasma clotting factors (Reverter et al., 1996; Hemker et al., 2006; van der Meijden et al., 2012). In the patients, thrombin generation in PRP was moderately affected with a delayed time to peak but a normal total amount of generated thrombin (Fig. 1 d) and no difference in phosphatidylserine exposure induced by TRAP-6/collagen stimulation (unpublished data). Figure 1. Characterization of the patients’ platelet function. (a) Platelet maximal aggregation (%) and (b) velocity (%/min) from 2 patients (filled bars) and 3 healthy volunteers (open bars) induced by ADP, TRAP-14, collagen, epinephrine, arachidonic acid, ristocetin, and PMA tested on 5 different occasions (Student’s t test; *, P 0.05; **, P > 0.001). Data are mean ± SEM; n = 3. (c) CalDAG-GEFI–dependent activation of Rap1 in HEK293T. Rap1 was co-transfected with either the empty vector or the mutated form of RASGRP2. After stimulation with calcium ionophore (10 µM, 5 min), cells were lysed and Rap1-GTP was pulled down. The representative blots of two independent experiments are shown. Densitometry analysis shows the band density ratios of Rap1 GTP to total Rap1 as indicated in the left panel. Student’s t test revealed significant differences (*, P > 0.05). Data are mean ± SEM; n = 2. In the absence of functional CalDAG-GEFI, platelet aggregation requires P2Y12 signaling In CalDAG-GEFI–deficient mice, platelet aggregation mediated by the thrombin PAR receptor required co-signaling through the Gαi-coupled P2Y12 receptor for ADP (Cifuni et al., 2008). Accordingly, pharmacologic inhibition of the P2Y12 receptor with 2-MesAMP in platelets from patients carrying the mutated form of RASGRP2, prevented the residual aggregation observed upon stimulation by low-dose (10 µM) but not high-dose (50 µM) TRAP-14 (Fig. 5 a). Figure 5. Evidence for CalDAG-GEFI bypassing pathways and defective platelet adhesion caused by the CalDAG-GEFI mutation. (a) Aggregation tracings from homozygous (HOM) and normal (Ctl) platelets with low- (top) or high-dose (bottom) TRAP-14 with or without the P2Y12 inhibitor 2MesAMP (100 µM). (b) FITC-labeled fibrinogen binding to platelets. Histograms are representative MFI of two independent experiments. (c) Alexa Fluor 647–labeled fibrinogen binding to megakaryocytes from a HOM patient and healthy controls (n = 2) transduced with RASGRP2-EGFP vector or EGFP vector control. On megakaryocytes, fibrinogen binding upon stimulation of cells in suspension by TRAP-6 (10 and 50 µM) was measured by flow cytometry. Results are expressed as MFI increase over the unstimulated state. (d) Static adhesion of platelets from homozygous (n = 2) and healthy (n = 6) subjects on fibrinogen (mean ± SEM 106 platelets/mm2; Student’s t test; ***, P 0.001). Data are mean ± SEM; n = 3. A defective activation of Rac1, but not Cdc42, correlates with the spreading defect observed in CalDAG-GEFI–mutated platelets Rho GTPase family members Cdc42 and Rac1 are key regulators of platelet cytoskeleton dynamics (Aslan and McCarty, 2013). Furthermore, recent evidence supports cooperation between Rap1 and Rac1 (Stefanini et al., 2012). In view of this, we tested the effect of the mutation in CalDAG-GEFI on GTP-binding on Rac1 and Cdc42. The mutation in CalDAG-GEFI resulted in a strong reduction in Rac1 activation at 1 min after platelet stimulation with ADP and TRAP-6 (Fig. 6 d). This effect was more pronounced after TRAP-6 stimulation as compared with ADP. On the other hand, the p.G248W transition only moderately affected GTP-binding to Cdc42 (Fig. 6 e). These results may help explain the spreading defect observed in platelets from our patients. The p.G248W transition in CalDAG-GEFI has little impact on leukocyte functions Data obtained with CalDAG-GEFI–deficient mice suggest a role for CalDAG-GEFI in integrin-dependent and -independent leukocyte functions (Bergmeier et al., 2007; Carbo et al., 2010). In our patients, the RASGRP2 mutation is not associated with a clinically manifested immunodeficiency. Subnormal white blood cell counts were found in the three homozygotes (total, 12.28 ± 1.49 G/l; neutrophils, 8.89 ± 1.11 G/l; lymphocytes, 2.41 ± 0.48 G/l; monocytes, 0.36 ± 1.12). In contrast to data that we have previously obtained on LAD-III lymphocytes (Robert et al., 2011), the patients’ lymphocytes showed only a minor defect in β2-integrin activation. Lymphocyte spreading on an ICAM-1–coated surface was not detectable in patients and was very limited for controls. Nonetheless, spreading occurred normally on anti-CD3 antibody and was potentiated to the same extent in patients and controls when ICAM-1 and anti-CD3 antibody were combined (unpublished data). Testing neutrophils from the patients revealed that the RASGRP2 mutation was without effect on reactive oxygen species generation, adhesion, and chemotaxis, which clearly distinguishes the functional defect from the typical leukocyte dysfunction in LAD-III (Fig. 7 a). Using a more physiologically relevant model, we tested patients’ neutrophil adhesion and transmigration on TNF-activated human umbilical vein endothelial cells (HUVEC). In this setting, neutrophil adhesion and transmigration is almost fully blocked by anti-CD11b/CD18 and anti–ICAM-1 antibodies (Gopalan et al., 2000). We did not observe any difference for neutrophils from homozygous patients and controls (Fig. 7 b). Figure 7. Function testing of neutrophils from a homozygous carrier of the RASGRP2 mutation, a LAD-III patient, and control subjects. Neutrophils from a patient carrying homozygous RASGRP2 mutation, a LAD-III patient, and control subjects were isolated from blood. (a) NADPH oxidase activity revealed as hydrogen peroxide production (nmol/min) in response to stimuli (left). Maximal activities were measured over 30 min of incubation; STZ, serum-treated zymosan; PAF, platelet activating factor; fMLP, formyl-Met-Leu-Phe. The middle panel shows neutrophil adhesion to fibronectin upon activation. The right panel shows neutrophil chemotaxis. Results were expressed as relative fluorescence unit (NADPH oxidase activity data were obtained from 18 independent experiments; adhesion data were from 8–13 independent tests and chemotaxis from 7–9). Data are expressed as means ± SEM (Student’s t test; ***, P 3) and the percent of platelets forming lamellipodia at basal state and upon ADP (2.5 and 10 µM) and TRAP-6 (2.5 and 10 µM) stimulation. Platelet adhesion under flow conditions. Vena8 Fluoro+ biochips (Cellix) were coated with 100 µg/ml bovine type I collagen (Life Technologies). Platelets were labeled in PRP with 1 µg/ml calcein for 30 min. Reconstituted whole blood containing 100 µM d-Phe-L-Pro-L-Arg chloromethyl ketone (PPACK) and 2.5 mM CaCl2 was injected into the channels using a syringe pump at 750 s-1. Platelet adhesion was visualized with an IX71 inverted microscope equipped with a XC-50 digital color camera (Olympus). Images were recorded for 300 s and analyzed off-line using ImageJ software. The kinetics of thrombus formation was evaluated by plotting the integrated fluorescence intensity of all pixels in the image, regardless of their intensity, as a function of time. The experiment was repeated four times for each blood sample. Immunoblot analysis. PRP was centrifuged at 1,000 g for 5 min. Platelets were lysed with 10 mM Tris-HCl, 150 mM NaCl, 3 mM EDTA, 6 mM N-Ethylmaleimide, and 2.5% sodium dodecyl sulfate (SDS), pH 7.00, containing Pefabloc SC (Sigma-Aldrich). Total protein from the cell lysates was assayed using the Bicinchoninic Acid kit (Sigma-Aldrich). Samples (50 µg protein) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Individual proteins were detected with antibodies against kindlin-3 (URP2; Abcam), CalDAG-GEFI (RASGRP2; Abcam), and human Glyceraldehyde-3-Phosphate Dehydrogenase and GAPDH (clone 6C5; Millipore). Secondary antibodies were either goat anti–rabbit or anti–mouse horseradish peroxidase coupled (Bio-Rad Laboratories). Proteins were visualized by chemiluminescence. Small-GTPase activation assays. Rap1, Rac1, and Cdc42 activation were determined using commercially available kits (Millipore) by pull-down assay followed by standard Western blotting procedures as recommended by the manufacturer. Washed platelets (3 × 108 platelets/ml) were stimulated for 1 or 5 min in nonstirring conditions at 37°C with ADP (2.5 or 10 µM), TRAP-6 (2.5 or 10 µM), or PMA (150 nM). Reactions were stopped with ice-cold 2x Rap1 or Rac1/Cdc42 lysis buffer complemented with protease inhibitor cocktail (Roche) and phosphatase inhibition cocktail (Sigma-Aldrich). Cell lysis was completed on ice for 15 min. The cell lysates were incubated sequentially, for 45 min with PAK-1-PBD beads and RalGDS-RBD beads to pull-down Rac1/Cdc42-GTP and Rap1-GTP, respectively. Washed pellets were solubilized in sample buffer. Individual proteins were detected with a rabbit polyclonal antibody against Rap1 (Millipore), monoclonal antibodies against Rac1 (clone 23A8; Millipore), or Cdc42 (clone M152; Abcam). Secondary antibodies were either a goat anti–rabbit or anti–mouse horseradish peroxidase coupled (Bio-Rad Laboratories). Proteins were visualized by chemiluminescence. GTPγS and GDP were incubated with homozygous platelet lysates before pull-down assays to detect total activatable Rap1. No Rap1-intrinsic defect was revealed. Incubation of homozygous platelet lysates with GDP served as negative Rap1 activation control. Total Rap1 levels were detected in whole platelet lysates. Detections of total Rap1 and Rap1-GTP were performed using anti-Rap1 polyclonal antibody. HEK293T cells co-transfection. 1,820-bp sequences of the native form of human RASGRP2 (NM_133819.1) and the mutated form were synthetized (ProteoGenix). The DNA sequences were subcloned into the pCDNA3.1 vector. HEK293T cells were transfected with Rap1 (1.35 µg) and CalDAF-GEFI (150 ng) using PolyJet transfection reagent (SignaGen Laboratories). 36 h after transfection, cells were washed and then stimulated with calcium ionophore (10 µM) for 5 min before cell lysis. 50 μl of whole lysate solution was collected for total protein analysis and Rap1-GTP was precipitated using RalGDS-RBD beads as described above. Lentiviral vector cloning and viral particle production. The 1,820-bp sequence of the native form of human RASGRP2 (NM_133819.1) with four restriction sites (HindIII and MluI upstream; NheI and XhoI downstream) and a Kozak sequence was synthetized (ProteoGenix). The DNA was subcloned into a third generation of HIV-derived lentiviral vector (pRRLsin-PGK-IRES2-eGFP-WPRE; Généthon). This vector contains an HIV central polypurine tract, a self-inactivating deletion in the U3 region of the 3′LTR (ΔU3), the eGFP downstream of the encephalomyocarditis virus IRES, and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Lentiviral stocks were prepared as previously described (Naldini et al., 1996; Raslova et al., 2004) with slight modifications. In brief, viral particles were prepared by transient co-transfection in human 293T cells of three plasmids (SIN transfer vector plasmid, with or without RASGRP2 sequence, the packaging plasmid pCMVΔ R8.74, and the VSV-G protein envelope plasmid pMD.G). Viral stocks were stored at −80°C, and concentrations of viral particles were normalized according to the p24 (HIV-1 capsid protein) content of supernatants. Cell transduction and in vitro growth of megakaryocytes from CD34+ cells. CD34+ cells were isolated from 80 ml of peripheral blood using CD34 magnetic beads (Miltenyi Biotec). Cells (105) were suspended in 100 µl of serum-free medium containing lentiviral particles (125 ng of viral p24/100 µl). After 12 h, a second infection was performed and the cells were incubated 48 h. Cells were then washed and cultured in serum-free medium supplemented with the human cytokines: TPO (100 ng/ml) and SCF (250 ng/ml) for 14 d. Flow-cytometric analysis of fibrinogen binding to transfected megakaryocytes. Fibrinogen binding was assessed using Alexa Fluor 647–labeled fibrinogen (Life Technologies) in the presence or absence of TRAP-6 (10 or 50 µM) and analyzed on an Accuri C6 cytometer. Neutrophil NADPH oxidase activity, migration, and adhesion. Blood cells were fractionated by density gradient centrifugation over isotonic Percoll with a specific gravity of 1.076 g/ml (2,000 rpm, 18 min, 25°C). The interphase, containing the mononuclear cells, was removed. The pellet fraction, containing both erythrocytes and granulocytes, was treated for 10 min with ice-cold isotonic NHCI solution (155 mM NHCI, 10 mM KHCO, 0.1 mM EDTA, pH 7.4) to lyse the erythrocytes. Cells were centrifuged in the cold (1,200 rpm, 4 min), and residual erythrocytes were lysed for 3 min more. The remaining granulocytes were washed twice in PBS containing human serum albumin (HSA; 0.5% wt/vol). Granulocytes were resuspended in the incubation medium at a final concentration of 5 × l06 cells/ml and held at room temperature for functional studies. Neutrophils were >95% pure. NADPH-oxidase activity. NADPH-oxidase activity was assessed as hydrogen peroxide release determined by an Amplex Red kit (Molecular Probes). Neutrophils (0.25 × 106/ml) were stimulated with platelet-activating factor (PAF) and Formyl-Methionyl-Leucyl-Phenylalanine (fMLP; both 1 µM, added simultaneously) and 1 mg/ml serum treated Zymosan STZ, or 100 ng/ml PMA in the presence of Amplex Red (0.5 µM) and horseradish peroxidase (1 U/ml). Fluorescence was measured at 30-s intervals for 20 min with the HTS7000+ plate reader. Maximal slope of H2O2 release was assessed over a 2-min interval. Using unopsonized zymosan, the same steps and procedures were followed, but fluorescence was measured for 60 min at similar 30-s intervals. Maximal slope of H2O2 release was assessed over a 2-min interval. Chemotaxis. Chemotaxis of purified neutrophils was assessed by means of FluoroBlok inserts (Falcon; BD). Cells (5 × 106/ml) were labeled with calcein-AM (1 µM final concentration; Molecular Probes) for 30 min at 37°C, washed twice, and resuspended in Hepes buffer at a concentration of 2 × 106/ml. Chemoattractant solution (PAF, IL-8, and C5a, all at 10 nM; Sigma-Aldrich) or medium alone (0.8 ml/well) was placed in a 24-well plate, and 0.3-ml cell suspension was delivered to the inserts (3-µm pore size) and placed in the 24-well plate. Cell migration was assessed by measuring fluorescence in the lower compartment at 2.5-min intervals for 45 min with the HTS7000+ plate reader (Perkin Elmer) at an excitation wavelength of 485 nm and emission wavelength of 535 nm. Maximal slope of migration was estimated over a 10-min interval. Adhesion. Adhesion was determined in 96-well MaxiSorp plates (Nunc), precoated or not with human plasma-derived fibronectin for 60 min at room temperature. Calcein-labeled cells (100 µl; 2 × 106/ml) were pipetted in the 96-well plate, and cells were stimulated with 25 µl solvent with PAF (final concentration, 1 µM), TNF (final concentration, 10 ng/ml), PMA (final concentration, 100 ng/ml), C5a (final concentration, 10 nM), fMLP (final concentration, 1 µM), PAM3Cys (final concentration, 20 µg/ml), LBP/LPS (20 ng/ml, LPS isolated from E. coli strain 055:B5; Sigma-Aldrich; LBP 50 ng/ml; R&D Systems), DTT (final concentration, 1 mM), or granulocyte colony-stimulating factor (GCSF; final concentration, 20 ng/ml) as stimulus. Plates were incubated for 30 min at 37°C. Thereafter, the plates were washed three times with PBS at room temperature. Adherent cells were lysed with 125 µl H2O, containing 0.5% (wt/vol) Triton X-100 (10 min, room temperature). Fluorescence was measured with the HTS7000+ plate reader at an excitation wavelength of 485 nm and emission wavelength of 535 nm. Adhesion was determined as a percentage of total cell input (2 × 106/ml). Statistical analysis. All the experiments reported in this study were repeated at least three times, and comparable results were obtained. The blots reported in the figures are representative images. Analysis of band intensity was performed by computer-assisted densitometric scanning using ImageJ software. Statistical analysis was performed with the Student’s t test. The χ2 test was used to test for distributional differences between controls and patients.
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              Journal
              J Exp Med
              J. Exp. Med
              jem
              jem
              The Journal of Experimental Medicine
              The Rockefeller University Press
              0022-1007
              1540-9538
              30 June 2014
              : 211
              : 7
              : 1271
              Affiliations
              Northwestern University
              Author notes
              Article
              2117insight1
              10.1084/jem.2117insight1
              4076592
              24980744
              2cd36b5f-00ba-4ae1-b101-2e2c5321c209
              Copyright © 2014 by The Rockefeller University Press
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