[Show abstract][Hide abstract] ABSTRACT: The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet's ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis.
[Show abstract][Hide abstract] ABSTRACT: Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use two distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized Candida albicans was found to be dependent on Complement Receptor 3 (CR3), the signaling proteins phosphatidylinositol-3-kinase (PI3K) and CARD9, but was independent of NADPH oxidase activity. The second mechanism for the killing of opsonized Candida albicans was strictly dependent on Fc-gamma receptors, protein kinase C (PKC) and Reactive Oxygen Species (ROS) production by the NADPH oxidase system. Each of the two pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensible for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency or Chronic Granulomatous Disease (CGD).
[Show abstract][Hide abstract] ABSTRACT: Objectives Aicardi–Goutières syndrome (AGS) is an autoimmune disorder that shares similarities with systemic lupus erythematous. AGS inflammatory responses specially target the cerebral white matter. However, it remains uncertain why the brain is the most affected organ, and little is known about the presence of autoantibodies in AGS. Here, we aim to profile specific autoantibodies in AGS and to determine whether these autoantibodies target cerebral epitopes.
Methods Using a multiplex microarray, we assessed the spectrum of serum autoantibodies in 56 genetically confirmed patients with AGS. We investigated the presence of immunoglobulins in AGS brain specimens using immunohistochemistry and studied the reactivity of sera against brain epitopes with proteomics.
Results Serum from patients exhibited high levels of IgGs against nuclear antigens (gP210, Nup62, PCNA, Ro/SSA, Sm/RNP complex, SS-A/SS-B), components of the basement membrane (entactin, laminin), fibrinogen IV and gliadin. Upon testing whether antibodies in AGS could be found in the central nervous system, IgGs were identified to target in vivo endothelial cells in vivo and astrocytes in brain sections of deceased patients with AGS. Using a proteomics approach, we were able to confirm that IgGs in serum samples from AGS patients bind epitopes present in the cerebral white matter.
Conclusions Patients with AGS produce a broad spectrum of autoantibodies unique from other autoimmune diseases. Some of these autoantibodies target endothelial cells and astrocytes in the brain of the affected patients, perhaps explaining the prominence of neurological disease in the AGS phenotype.
Annals of the Rheumatic Diseases 06/2014; · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE. The aim of this study in clinically active juvenile idiopathic arthritis (JIA) was to assess the frequency and distribution pattern of synovitis as hallmark of disease and additional soft-tissue and bony abnormalities on MRI in the knee and wrist as two target joints. MATERIALS AND METHODS. MRI datasets of 153 clinically active JIA patients (110 with knee and 43 with wrist involvement) were evaluated independently by two readers for the presence of literature-based imaging features: "synovial hypertrophy," "bone marrow changes," "bone erosions," "tenosynovitis" (only in the wrist), and "cartilage lesions" (only in the knee) in accordance with validated definitions and scoring locations. RESULTS. Synovial hypertrophy was most frequently observed-both in the knee and in the wrist (61.8-65.1% of cases). For the knee, the most frequently involved locations were the cruciate ligaments (46/183 locations [25.1%] affected with synovial hypertrophy) and medial patella (18/62 locations [29.0%] with bone marrow changes). Cartilage lesions and bone erosions were rare (5.5-7.3% of cases). For the wrist, most frequently involved were the radiocarpal joint (21/64 locations [32.8%] with synovial hypertrophy), lunate (7/46 locations [15.2%] with bone marrow changes), and capitate or triquetrum (6/28 locations [21.4%] with bone erosions). Tenosynovitis was a common wrist-specific feature (46.5% of cases). MRI showed no abnormalities in a subgroup of patients with clinically active knee (23.6%) and wrist (16.3%) involvement. CONCLUSION. The distribution pattern of MRI abnormalities in the knee and wrist of active JIA patients provides a practical tool to detect a signature of JIA disease activity in target joints.
American Journal of Roentgenology 05/2014; 202(5):W439-46. · 2.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To compare DCE-MRI parameters and the relative number of time-intensity curve (TIC) shapes as derived from pixel-by-pixel DCE-MRI TIC shape analysis between knees of clinically active and inactive juvenile idiopathic arthritis (JIA) patients.
DCE-MRI data sets were prospectively obtained. Patients were classified into two clinical groups: active disease (n = 43) and inactive disease (n = 34). Parametric maps, showing seven different TIC shape types, were created per slice. Statistical measures of different TIC shapes, maximal enhancement (ME), maximal initial slope (MIS), initial area under the curve (iAUC), time-to-peak (TTP), enhancing volume (EV), volume transfer constant (K (trans)), extravascular space fractional volume (V e) and reverse volume transfer constant (k ep) of each voxel were calculated in a three-dimensional volume-of-interest of the synovial membrane.
Imaging findings from 77 JIA patients were analysed. Significantly higher numbers of TIC shape 4 (P = 0.008), median ME (P = 0.015), MIS (P = 0.001) and iAUC (P = 0.002) were observed in clinically active compared with inactive patients. TIC shape 5 showed higher presence in the clinically inactive patients (P = 0.036).
The pixel-by-pixel DCE-MRI TIC shape analysis method proved capable of differentiating clinically active from inactive JIA patients by the difference in the number of TIC shapes, as well as the descriptive parameters ME, MIS and iAUC.
• The pixel-by-pixel TIC shape method differentiates clinically active and inactive JIA patients • Significantly higher numbers of TIC shape 4 were observed in clinically active patients • DCE-MRI parameters ME, MIS and iAUC differ between active and inactive patients • The pixel-by-pixel analysis method allows direct visualization of the heterogeneously distributed disease • The DCE-MRI TIC shape method may serve as a quantitative outcome measure.
[Show abstract][Hide abstract] ABSTRACT: We present a case of a fatal Epstein-Barr infection in a 17-year-old male patient suspected to be caused by X-linked lymphoproliferative disease. At the time of hospitalization, DNA diagnostics was not available. The suspected diagnosis was confirmed several years later when a SH2D1A missense mutation was identified in stored patient DNA. Extended pedigree analysis showed that this mutation occurred de novo in his mother. In addition, we provide a summary of the currently listed SH2D1A mutations. Conclusion: This case report underlines the importance of DNA storage, pedigree analysis, and multidisciplinary care in patients with rare diseases and their families.
European Journal of Pediatrics 04/2014; · 1.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Kawasaki disease (KD) is an acute paediatric vasculitis. The psychosocial consequences of this sudden illness for parents are unknown. This study aimed to evaluate health related quality of life (HRQOL) and parental perceptions of child vulnerability (PPCV) in parents of children with KD, and to identify variables associated with PPCV.
This cross-sectional study included 288 parents (83% mothers) of KD patients (mean age 8.7 years). HRQOL was assessed using the TNO-AZL Questionnaire for Adult's HRQOL (TAAQOL) and PPCV using the Child Vulnerability Scale (CVS). Scores of KD parents were compared with reference groups of Dutch parents. Logistic regression analyses were performed to examine associated variables.
The HRQOL of KD parents was comparable to the HRQOL of parents of healthy children. However, KD parents showed significantly higher PPCV, regarding both the median CVS total score and the percentage in the clinical range. No differences were found in CVS outcomes between KD parents and parents of a chronically ill child. None of the studied parental, child and disease characteristics were significantly associated with PPCV.
Parents perceived their KD child more vulnerable to illness than healthy children, while in reality the majority had fully recovered from KD. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Human blood neutrophils normally express two FcγRs (FcγRIIa and FcγRIIIb) that, upon multivalent binding of IgG in immune complexes or on opsonized targets, mediate responses such as phagocytosis, Ab-dependent cellular cytotoxicity, and respiratory burst. Allelic variants have been described for both FcγRIIa (131H/R) and FcγRIIIb (NA1/NA2/SH), with different binding affinity for IgG subclasses. Because neither of these variants acts alone, we have set out to systematically analyze in a large cohort of healthy FCGR2/3-genotyped volunteers how the different haplotypes of neutrophil FcγRs functionally interact. Maximal IgG-induced H2O2 production by neutrophils from individuals with different (homozygous) haplotypes was observed in the following order: 131HH-NA2NA2 > 131RR-NA1NA1 > 131HH-NA1NA1 > 131RR-NA2NA2. Although FcγRIIa 131H is known to bind IgG1 and IgG2 more avidly, no such differences in affinity are known for FcγRIIIb variants. Nonetheless, a remarkable impact of the FcγRIIIb variants on IgG-mediated neutrophil activity was thus demonstrated, which was not explained by differences in FcγR surface expression. The FcγR expression profile was changed by overnight G-CSF/IFN-γ activation of the neutrophils and eliminated any haplotypic impact on the respiratory burst. To our knowledge, our results are the first to provide an integrated functional analysis of neutrophil FcγR haplotypes and suggest that particularly the early phase of IgG-mediated neutrophil reactivity is influenced by FCGR2/3 genotypic variation.
The Journal of Immunology 02/2014; · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retrotransposon-mediated insertion of a LINE-1 or an Alu element into a human gene is a well known pathogenic mechanism. We report a novel LINE-1-mediated insertion of a transcript from the TMF1 gene on chromosome 3 into the CYBB gene on the X chromosome. In a Dutch male patient with chronic granulomatous disease a 5.8-kb, incomplete and partly exonized TMF1 transcript was identified in intron 1 of CYBB, in opposite orientation to the host gene. The sequence of the insertion showed the hallmarks of a retrotransposition event, with an antisense polyA tail, target site duplication and a consensus LINE-1 endonuclease cleavage site. This insertion induced aberrant CYBB mRNA splicing, with inclusion of an extra 117-bp exon between exons 1 and 2 of CYBB. This extra exon contained a premature stop codon. The retrotransposition took place in an early stage of fetal development in the mother of the patient, because she showed a somatic mosaicism for the mutation that was not present in the DNA of her parents. However, the mutated allele was not expressed in the patient's mother because the insertion was found only in the methylated fraction of her DNA. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Autosomal dominant Hyper IgE syndrome (AD-HIES) is a rare and complex primary immunodeficiency that affects multiple systems. Mutations in signal transducer and activator of transcription 3 (STAT3) gene cause AD-HIES. These mutations have a dominant-negative effect and the presence of such mutations is associated with a clinical phenotype. We aim to describe genetic and clinical characteristics of patients with AD-HIES in our clinic and to highlight the variability of clinical patterns in the same family.
We describe six patients, four individuals of the same family and two unrelated patients. All patients were given a clinical score based on disease phenotype according to the National Institute of Health (NIH) score. Mutation analysis of STAT3 was done by PCR amplification of all coding exons followed by bidirectional sequencing using the BigDye kit v1.1 and an ABI3700 genetic analyzer (Applied Biosystems).
All six patients had DNA binding region point mutations: a proband and his three children with p.Phe384Leu mutation, a patient with p.Arg382Trp substitution and a patient with p.Arg382Gln mutation. All of these mutations were previously reported. Patients differed in infectious, immunologic and somatic features. We observed an extreme variability in disease phenotype within the reported family with one genetically affected patient displaying an 'unaffected' phenotype.
Although the genetic cause of AD-HIES is known, more studies are required to better understand the possible additional factors that may affect disease expressivity within families and the clinical diversity of the disease.
Journal of Clinical Immunology 01/2014; · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the incidence, disease presentation, treatment and cardiac outcome of Kawasaki disease (KD) in the Netherlands.
The national Dutch Pediatric Surveillance Unit (DPSU) was used to prospectively register new KD cases from 2008 through 2012. Questionnaires were sent to pediatricians to obtain clinical information.
Nationwide 341 cases were reported during the 5-year study period, of which 319 questionnaires (93.0%) were returned. The mean incidence of KD was estimated to be 5.8/100,000 children <5 years of age. The median age at disease onset was 2.4 years (range 0.1-14.6 years) and 79.2% of cases were <5 years of age). The male to female ratio was 1.5 to 1. Incomplete KD was diagnosed in 22.3% of cases and these cases were significantly younger than complete cases (median, 1.1 [0.1-13.7] versus 2.8 [0.2-14.6] years, p<0.001). In total, 308 patients (96.6%) received IVIG. Retreatment with IVIG was given in 71 (23.1%), and additional steroid treatment in 17 patients (5.5%). During the acute phase coronary artery aneurysms (CAA) developed in 43 cases (13.5%). Multivariate logistic regression analysis showed that male gender, delay of treatment (>10 days) and IVIG retreatment were independent risk factors for CAA development.
This prospective study of KD in the Netherlands revealed a mean annual incidence of 5.8/100,000 children <5 years of age. Clinicians should consider the diagnosis of KD in young (male) children with persistent inexplicable fever to start IVIG treatment within 10 days to prevent development of CAA.
The Pediatric Infectious Disease Journal 01/2014; · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sepsis is a severe and life-threatening systemic inflammatory response to infection that affects all populations and age groups. The pathophysiology of sepsis is associated with aberrant interaction between leukocytes and the vascular endothelium. As inflammation progresses, the adhesion molecules that mediate these interactions become shed from cell surfaces and accumulate in the blood as soluble isoforms that are being explored as potential prognostic disease biomarkers. We critically review the studies that have tested the predictive value of soluble adhesion molecules in sepsis pathophysiology with emphasis on age, as well as the underlying mechanisms and potential roles for inflammatory shedding. Five soluble adhesion molecules are associated with sepsis, specifically, E-selectin, L-selectin and P-selectin, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. While increased levels of these soluble adhesion molecules generally correlate well with the presence of sepsis, their degree of elevation is still poorly predictive of sepsis severity scores, outcome and mortality. Separate analyses of neonates, children and adults demonstrate significant age-dependent discrepancies in both basal and septic levels of circulating soluble adhesion molecules. Additionally, a range of both clinical and experimental studies suggests protective roles for adhesion molecule shedding that raise important questions about whether these should positively or negatively correlate with mortality. In conclusion, while predictive properties of soluble adhesion molecules have been researched intensively, their levels are still poorly predictive of sepsis outcome and mortality. We propose two novel directions for improving clinical utility of soluble adhesion molecules: the combined simultaneous analysis of levels of adhesion molecules and their sheddases; and taking age-related discrepancies into account. Further attention to these issues may provide better understanding of sepsis pathophysiology and increase the usefulness of soluble adhesion molecules as diagnostic and predictive biomarkers.
Critical care (London, England) 01/2014; 18(1):204. · 4.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myeloid antigen-presenting cells (APCs) tailor immune responses to the pathogen involved through the production of specific pro- and anti-inflammatory cytokines. It is becoming increasingly clear that the ultimate cytokine profile produced by myeloid APCs crucially depends on interaction between multiple pathogen recognizing receptors. In this respect, we recently identified an important role for cross-talk between Fc gamma receptor IIa (FcγRIIa) and Toll-like receptors (TLRs) in human dendritic cells (DCs), which induces anti-bacterial immunity through the selective induction of TNFα and Th17-promoting cytokines. Here, we show that FcγRIIa-TLR cross-talk is not restricted to DCs, but is a common feature of various human myeloid APC subsets including monocytes and macrophages. Interestingly, FcγRIIa-TLR cross-talk in monocytes resulted in the induction of a cytokine profile distinct from that in DCs and macrophages, indicating that FcγRIIa stimulation induces cell-type and tissue specific responses. Surprisingly, we show that the FCGR2A H131R single nucleotide polymorphism (SNP), which is known to greatly affect FcγRIIa-mediated uptake of IgG2-opsonized bacteria, did not affect FcγRIIa-dependent cytokine production, indicating that these processes are differently regulated. In addition, we demonstrate that FcγRIIa selectively synergized with TLRs, IL-1R, and IFNγR, but did not affect cytokine production induced by other receptors such as C-type lectin receptor Dectin-1. Taken together, these data demonstrate that FcγRIIa-dependent modulation of cytokine production is more widespread than previously considered, and indicate that cross-talk of FcγRIIa with various receptors and in multiple cell types contributes to the induction of pathogen and tissue-specific immunity.
[Show abstract][Hide abstract] ABSTRACT: A chronic illness, such as Juvenile Idiopathic Arthritis (JIA), has an impact on the whole family, especially on parents caring for the ill child. Therefore the aim of this study is to evaluate parental Health Related Quality of Life (HRQOL) and parental perceptions of child vulnerability (PPCV) and associated variables in parents of a child with JIA.
[Show abstract][Hide abstract] ABSTRACT: MBL-deficiency is a commonly occurring deficiency of the innate immune system, affecting a substantial part of the population and has been extensively studied. MBL appears to function as a disease modifier. The role of MBL in different conditions is context-dependent. Many clinical studies show conflicting results, which can be partially explained by different definitions of MBL-deficiency, including phenotype- and genotype-based approaches. In this review we give an overview of literature of MBL, its role in different pathologies, diseases and patient populations. We review MBL replacement studies, and discuss the potential of MBL substitution therapy. We finally suggest that new MBL substitution trials should be conducted within a predefined patient population. MBL-deficiency should be based on serum levels and confirmed by genotyping.
[Show abstract][Hide abstract] ABSTRACT: MBL-deficiency has been associated with an increased frequency and severity of infection, in particular in children and under immunocompromized conditions. In an open uncontrolled safety and pharmacokinetic MBL-substitution study using plasma-derived MBL (pdMBL) in MBL-deficient pediatric oncology patients, we found that despite MBL trough levels above 1.0μg/ml MBL functionality was not efficiently restored upon ex vivo testing. PdMBL showed C4-converting activity by itself, indicating the presence of MASPs. Upon incubation of pdMBL with MBL-deficient sera this C4-converting activity was significantly reduced. Depletion of the MASPs from pdMBL, paradoxically, restored the C4-converting activity. Subsequent depletion or inhibition of C1-inh, the major inhibitor of the lectin pathway, in the recipient serum restored the C4-converting activity as well. Complexes between MBL/MASPs and C1-inh (MMC-complexes) were detected after ex vivo substitution of MBL-deficient serum with pdMBL. These MMC-complexes could also be detected in the sera of the patients included in the MBL-substitution study shortly after pdMBL infusion. Altogether, we concluded that active MBL-MASP complexes in pdMBL directly interact with C1-inh in the recipient, leading to the formation of a multimolecular complex between C1-inh and MBL/MASPs, in contrast to the classical pathway where C1r and C1s are dissociated from C1q by C1-inh. Because of the presence of activated MASPs in the current pdMBL products efficient MBL-mediated host protection cannot be expected because of the neutralizing capacity by C1-inh.