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Dirk Roos

Publications and source records attributed to Dirk Roos.

15 recordsLinked to original sources
PubMed2003

Functional epitope on human neutrophil flavocytochrome b558.

mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.

Antibodies, Monoclonal↗
PubMed2003

Apoptotic neutrophils in the circulation of patients with glycogen storage disease type 1b (GSD1b).

Glycogen storage disease type 1b (GSD1b) is a rare autosomal recessive disorder characterized by hypoglycemia, hepatomegaly, and growth retardation, and associated-for unknown reasons- with neutropenia and neutrophil dysfunction. In 5 GSD1b patients in whom nicotin-amide adenine dinucleotide phosphate-oxidase activity and chemotaxis were defective, we found that the majority of circulating granulocytes bound Annexin-V. The neutrophils showed signs of apoptosis with increased caspase activity, condensed nuclei, and perinuclear clustering of mitochondria to which the proapoptotic Bcl-2 member Bax had translocated already. Granulocyte colony-stimulating factor (G-CSF) addition to in vitro cultures did not rescue the GSD1b neutrophils from apoptosis as occurs with G-CSF-treated control neutrophils. Moreover, the 2 GSD1b patients on G-CSF treatment did not show significantly lower levels of apoptotic neutrophils in the bloodstream. Current understanding of neutrophil apoptosis and the accompanying functional demise suggests that GSD1b granulocytes are dysfunctional because they are apoptotic.

Adolescent↗
PubMed2003

The Rh complex exports ammonium from human red blood cells.

The Rh blood group system represents a major immunodominant protein complex on red blood cells (RBC). Recently, the Rh homologues RhAG and RhCG were shown to promote ammonium ion transport in yeast. In this study, we showed that also in RBC the human Rh complex functions as an exporter of ammonium ions. We measured ammonium import during the incubation of RBC in a solution containing a radiolabelled analogue of NH4Cl (14C-methyl-NH3Cl). Rhnull cells of the regulator type (expressing no Rh complex proteins) accumulated significantly higher levels (P = 0.05) of radiolabelled methyl-ammonium ions than normal RBC, at room temperature. Rhnull cells of the amorph type (expressing limited amounts of Rh complex proteins) accumulated an intermediate amount of methyl-ammonium ions. To show that decreased ammonium export contributes to its accumulation, the release of intracellular methyl-ammonium from the cells was measured over time. In 30 s, normal RBC released 87% of the intracellular methyl-ammonium ions, whereas Rhnull cells of the regulator type released only 46%. We conclude that the Rh complex is involved in the export of ammonium from RBC.

Benzylamines↗
PubMed2003

Expression of myeloperoxidase (MPO) by neutrophils is necessary for their activation by anti-neutrophil cytoplasm autoantibodies (ANCA) against MPO.

Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase-3 and myeloperoxidase (MPO) activate tumor necrosis factor-alpha-primed neutrophils in vitro. We used neutrophils from one completely and one partially MPO-deficient donor to assess the requirement of MPO expression for neutrophil activation by anti-MPO antibodies. The MPO deficiencies were defined enzymatically, by immunocytochemistry and by immunoblotting. The mutations in the MPO genes of these donors were identified as a combination of a novel splice-site mutation at the 3' end of intron 11 (A-2-->C), a deletion of 14 nucleotides in exon 9 (A1555-C1568), and a novel C1907 --> T (636Thr-->Met) substitution in exon 11 in the completely MPO-deficient donor and as the same splice-site mutation and a novel C995 --> T (332Ala-->Val) substitution in exon 7 in the partially MPO-deficient donor. Monoclonal antibody 4.15 against MPO and MPO-ANCA-immunoglobulin G induced no superoxide anion production in these MPO-deficient neutrophils despite a normal production induced by other stimuli. Thus, the presence of MPO is a conditio sine qua non for neutrophil activation by anti-MPO antibodies. Moreover, we demonstrated that by means of these MPO-deficient cells, hydrogen peroxide may diffuse from neutrophils to surrounding cells, which may contribute to the damage induced by oxygen radicals in the pathology of systemic vasculitides.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2002

Repression of rac2 mRNA expression by Anaplasma phagocytophila is essential to the inhibition of superoxide production and bacterial proliferation.

Anaplasma phagocytophila, the etiologic agent of human granulocytic ehrlichiosis, is an emerging bacterial pathogen that invades neutrophils and can be cultivated in HL-60 cells. Infected neutrophils and HL-60 cells fail to produce superoxide anion (O(2)(-)), which is partially attributable to the fact that A. phagocytophila inhibits transcription of gp91(phox), an integral component of NADPH oxidase. cDNA microarray and RT-PCR analyses demonstrated that transcription of the gene encoding Rac2, a key component in NADPH oxidase activation, was down-regulated in infected HL-60 cells. Quantitative RT-PCR demonstrated that rac2 mRNA expression was reduced 7-fold in retinoic acid-differentiated HL-60 cells and 50-fold in neutrophils following A. phagocytophila infection. Rac2 protein expression was absent in infected HL-60 cells. Rac1 and Rac2 are interchangeable in their abilities to activate NADPH oxidase. HL-60 cells transfected to express myc-tagged rac1 and gp91(phox) from the CMV immediate early promoter maintained the ability to generate O(2)(-) 120 h postinfection. A. phagocytophila proliferation was severely inhibited in these cells. These results directly attribute the inhibition of rac2 and gp91(phox) transcription to the loss of NADPH oxidase activity in A. phagocytophila-infected cells and demonstrate its importance to bacterial intracellular survival.

Anaplasma phagocytophilum↗
PubMed2002

Tumor necrosis factor alpha induces a caspase-independent death pathway in human neutrophils.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine with multiple roles in the immune system, including the induction and potentiation of cellular functions in neutrophils (PMNs). TNF-alpha also induces apoptotic signals leading to the activation of several caspases, which are involved in different steps of the process of cell death. Inhibition of caspases usually increases cell survival. Here, we found that inhibition of caspases by the general caspase inhibitor zVAD-fmk did not prevent TNF-alpha-induced PMN death. After 6 hours of incubation, TNF-alpha alone caused PMN death with characteristic apoptotic features (typical morphologic changes, DNA laddering, external phosphatidyl serine [PS] exposure in the plasma membrane, Bax clustering and translocation to the mitochondria, and degradation of mitochondria), which coincided with activation of caspase-8 and caspase-3. However, in the presence of TNF-alpha, PMNs died even when caspases were completely inhibited. This type of cell death lacked nuclear features of apoptosis (ie, no DNA laddering but aberrant hyperlobulated nuclei without typical chromatin condensation) and demonstrated no Bax redistribution, but it did show mitochondria clustering and plasma membrane PS exposure. In contrast, Fas-triggered PMN apoptosis was completely blocked by zVAD-fmk. Experiments with scavengers of reactive oxygen species (ROS) and with inhibitors of mitochondrial respiration, with PMN-derived cytoplasts (which lack mitochondria) and with PMNs from patients with chronic granulomatous disease (which have impaired nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) indicated that TNF-alpha/zVAD-fmk-induced cell death depends on mitochondria-derived ROS. Thus, TNF-alpha can induce a "classical," caspase-dependent and a "nonclassical" caspase-independent cell death.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Deletion of leucine 61 in glucose-6-phosphate dehydrogenase leads to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections.

In this study the blood cells of 4 male patients from 2 unrelated families with chronic nonspherocytic anemia and recurrent bacterial infections were investigated. The activity of glucose-6- phosphate dehydrogenase (G6PD) in the red blood cells and in the granulocytes of these patients was below detection level. Moreover, their granulocytes displayed a decreased respiratory burst upon activation. Sequencing of genomic DNA revealed a novel 3-base pair (TCT) deletion in the G6PD gene, predicting the deletion of a leucine at position 61. The mutant G6PD protein was undetectable by Western blotting in the red blood cells and granulocytes of these patients. In phytohemagglutinin-stimulated lymphocytes the G6PD protein was present, but the amount of G6PD protein was strongly diminished in the patients' cells. Purified mutant protein from an Escherichia coli expression system showed decreased heat stability and decreased specific activity. Furthermore, we found that the messenger RNA of G6PD(180-182delTCT) is unstable, which may contribute to the severe G6PD deficiency observed in these patients. We propose the name "G6PD Amsterdam" for this new variant.

Adolescent↗
PubMed2002

Evidence consistent with human L1 retrotransposition in maternal meiosis I.

We have used a unique polymorphic 3' transduction to show that a human L1, or LINE-1 (long interspersed nucleotide element-1), retrotransposition event most likely occurred in the maternal primary oocyte during meiosis I. We characterized a truncated L1 retrotransposon with a 3' transduction that was inserted, in a Dutch male patient, into the X-linked gene CYBB, thereby causing chronic granulomatous disease. We used the unique flanking sequence to localize the precursor L1 locus, LRE3, to chromosome 2q24.1. In a cell culture assay, the retrotransposition frequency of LRE3 is greater than that for any other element that has been tested to date. The patient's mother had two LRE3 alleles that differed slightly in the 3'-flanking genomic DNA. The patient had a single LRE3 allele that was identical to one of the maternal alleles; however, the patient's insertion matched the maternal LRE3 allele that he did not inherit. Other data indicate that there is only a small chance that the father (unavailable for analysis) carries the precursor LRE3 allele. In addition, paternal origin of the insertion would have required that an LRE3 mRNA transcribed before meiosis II be carried separately from its precursor LRE3 allele in the fertilizing sperm. Since the mother carries a potential precursor allele and the insertion was on the patient's maternal X chromosome, it is highly likely that the insertion originated during maternal meiosis I.

Adult↗
PubMed2002

Granulocyte colony-stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neutrophils.

The exact mechanism of apoptosis in neutrophils (PMNs) and the explanation for the antiapoptotic effect of granulocyte colony-stimulating factor (G-CSF) in PMNs are unclear. Using specific fluorescent mitochondrial staining, immunofluorescent confocal microscopy, Western blotting, and flow cytometry, this study found that PMNs possess an unexpectedly large number of mitochondria, which are involved in apoptosis. Spontaneous PMN apoptosis was associated with translocation of the Bcl-2-like protein Bax to the mitochondria and subsequent caspase-3 activation, but not with changes in the expression of Bax. G-CSF delayed PMN apoptosis and prevented both associated events. These G-CSF effects were inhibited by cycloheximide. The general caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-fmk) prevented caspase-3 activation and apoptosis in PMNs, but not Bax redistribution. PMN-derived cytoplasts, which lack a nucleus, granules, and mitochondria, spontaneously underwent caspase-3 activation and apoptosis (phosphatidylserine exposure), without Bax redistribution. zVAD-fmk inhibited both caspase-3 activation and phosphatidylserine exposure in cultured cytoplasts. Yet, G-CSF prevented neither caspase-3 activation nor apoptosis in cytoplasts, confirming the need for protein synthesis in the G-CSF effects. These data demonstrate that (at least) 2 routes regulate PMN apoptosis: one via Bax-to-mitochondria translocation and a second mitochondria-independent pathway, both linked to caspase-3 activation. Moreover, G-CSF exerts its antiapoptotic effect in the first, that is, mitochondria-dependent, route and has no impact on the second.

Adult↗
PubMed2002

Prenatal diagnosis in two families with autosomal, p47(phox)-deficient chronic granulomatous disease due to a novel point mutation in NCF1.

OBJECTIVE: Prenatal diagnosis was required in two unrelated families with chronic granulomatous disease (CGD) patients who lacked expression of p47(phox) protein; thus a search for mutations in NCF1 was undertaken. METHODS: Gene scanning was applied to establish the relative number of coding and pseudo-NCF1 genes. PCRs specific for coding NCF1 cDNA and coding NCF1 exon-7 genomic DNA were devised. RESULTS: The normal 1:2 ratio of coding and pseudo-NCF1 genes was found in the patients. Sequencing of the RT-PCR product specific for mRNA from the coding NCF1 genes revealed a novel homozygous G579A mutation in both patients, changing the TGG codon for Trp193 into the TAG stop codon. This mutation was confirmed in genomic DNA. The parents of both patients were found to be heterozygotes for this mutation. In the chorionic villus DNA of the first family a heterozygous G579A mutation was found. Postpartum, functional NADPH oxidase tests were normal. In the second family, this mutation was present in homozygous form in the chorionic villus DNA. Following termination of the pregnancy, the diagnosis of p47(phox)-deficient CGD was confirmed on DNA extracted from fetal blood. CONCLUSION: This is the first report of prenatal diagnosis in p47(phox)-deficient CGD.

Adolescent↗
PubMed2002

Genetic analysis of patients with leukocyte adhesion deficiency: genomic sequencing reveals otherwise undetectable mutations.

OBJECTIVE: The aim of this study was to analyze mutations in DNA from patients with leukocyte adhesion deficiency (LAD), an immunodeficiency caused by absence of the beta(2) subunit (CD18) of the leukocyte integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and CR4 (CD11d/CD18). METHODS: We developed genomic DNA PCR sequencing to detect mutations not only in exons but also in introns. RESULTS: Eight LAD patients were analyzed, of which five had homozygous mutations, i.e., a 0.8-kb deletion, a branchpoint mutation in intron 5 causing mRNA missplicing, a nonsense mutation, and two missense mutations. Four of these mutations are novel. We cotransfected the two mutant CD18 proteins with normal CD11a, b, or c in COS cells. This resulted in absence of all three beta(2) integrins on the surface of cells transfected with CD18(252Arg). However, CD18(593Cys) supported some LFA-1 and p150,95 formation in COS cells. The other three patients were compound heterozygotes in which only one allele had previously been characterized, because the other alleles were undetectable at the cDNA level. We identified the unknown mutations as a novel two-nucleotide deletion, a nonsense mutation, and a single nucleotide deletion. CONCLUSION: Our method allows identification of mutations in CD18 from genomic DNA. This opens the possibility of early prenatal diagnosis of LAD and reliable carrier detection.

Alleles↗
PubMed2002

Decreased redox state in red blood cells from patients with sarcoidosis.

BACKGROUND AND AIM OF THE WORK: The glutathione system has a key role in the defence against oxidative stress. To function properly, this system needs NADPH to maintain glutathione (GSH) in its reduced form. We hypothesized that the clinical problems associated with sarcoidosis might be related to a decreased anti-oxidant defence and we therefore measured the activity of the NADPH-generating enzyme glucose-6-phosphate dehydrogenase (G6PD), the GSH-regenerating enzyme glutathione reductase (GR) and indirectly the level of NADPH in red blood cells from patients with sarcoidosis. METHODS: In a population of sarcoidosis (n = 88) patients, G6PD, GR and GR activity after incubation with chromate (GR-Cr) were measured in erythrocytes. A decreased concentration of NADPH was revealed by an increased GR-Cr (> 0.6 IU/g Hb). To exclude a mutation in the G6PD gene, sequencing was performed in cases with an abnormal GR-Cr. Sarcoidosis pulmonary disease severity was evaluated by means of laboratory data, radiographic staging, HRCT scoring, pulmonary function and exercise capacity testing. RESULTS: Fourteen (29.2%) females and one (2.5%) male demonstrated an increased GR-Cr test, indicative of a decreased NADPH level. Patients with an abnormal test result demonstrated also significantly increased ACE and GR values (p < 0.05). Only one female case (of 6 tested) appeared to have a mutation in the G6PD gene. CONCLUSION: In a considerable percentage of female patients with sarcoidosis, a decreased level of NADPH in the erythrocytes was found.

Adolescent↗