New approaches to the detection of myeloperoxidase deficiency

Dri, Pietro; Cramer, Rita; Soranzo,; Comin, Alessandra; Miotti, Valeria; Patriarca, Pierluigi

doi:10.1182/blood.v60.2.323.323

Cited by 39 publications

(1 citation statement)

References 14 publications

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“…Neutrophils in the pellet were resuspended in phosphate-buffered saline (PBS) solution. Erythrocytes were lysed twice with a frozen hypotonic lysis buffer as described by Dri et al (33). Briefly, the lysis buffer contained 155 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA for a pH of 7.4.…”

Section: Isolation Of Human Neutrophilsmentioning

confidence: 99%

Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis

et al. 2021

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Reactive oxygen species (ROS) play a crucial role in the cellular defense against S. aureus, as evidenced by the importance of this pathogen in patients lacking the ROS-generating phagocyte NADPH oxidase NOX2. ROS concentrations required to kill S. aureus in vitro are much higher than those found in the phagosome. We therefore hypothesized that sublethal ROS concentrations may play a role in S. aureus gene dysregulation and investigated the in vitro transcriptomic response of S. aureus to sublethal concentrations of hydrogen peroxide (H2O2). A striking observation of these experiments was a coordinated and massive downregulation of genes involved in pyrimidine metabolism. Using transposon insertion mutants, we demonstrated that deletion of carA, a gene involved in pyrimidine synthesis, led to a significant growth defect and to an increased sensitivity of S. aureus to added H2O2. The phenotype of the carA mutant could be reversed through supplementation with the pyrimidine precursor uracil, or with a multicopy vector encoding carA. As opposed to the impact of ROS on extracellular survival, carA deletion did not affect the intracellular survival in neutrophils. Our results raise the possibility that ROS-dependent downregulation of pyrimidine metabolism might be a survival strategy of S. aureus, allowing colonization through intracellular survival, while decreasing the risk of killing the host through dampened extracellular growth.

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Section: Isolation Of Human Neutrophilsmentioning

confidence: 99%

Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis

et al. 2021

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Genetic characterization of myeloperoxidase deficiency in Italy

Marchetti¹,

Patriarca²,

Solero³

et al. 2004

Hum. Mutat.

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Hereditary myeloperoxidase (MPO) deficiency (MPOD) is the most common neutrophil biochemical defect, and is characterized by a lack of peroxidase activity. In order to extend the epidemiological studies on hereditary MPOD in Italy, a population screening was carried out to detect mutations in the MPO gene. Of approximately 40,000 individuals analyzed, seven partial and eight total MPO-deficient subjects were identified. The genetic characterization of the subjects showed the presence of three already-known mutations (c.752T>C, c.1705C>T, and c.1566_1579del14) and six novel mutations: four missense mutations (c.995C>T, c.1112A>G, c.1715T>G, and c.1927T>C), then a deletion of an adenine within exon 3 (c.325delA) and a mutation within the 3' splice site of intron 11 (c.2031-2A>C). The novel missense mutations cause the substitution of the residues p.A332V, p.D371G, p.L572W, and p.W643R, respectively, and the potential structural changes are discussed. The c.325delA deletion causes a shift of the reading frame with the occurrence of a premature stop codon within the propeptide. Then, considering the difficulty in obtaining bone marrow samples from MPO-deficient subjects to study MPO mRNA splicing in vivo, we set up an eukaryotic expression system to investigate how the c.2031-2A>C mutation alters the MPO pre-mRNA splicing. The activation of a cryptic 3' splice site located 109nt upstream of the authentic 3' splice site was observed. The 109nt-insertion causes a shift in the reading frame that should lead to the generation of an abnormal MPO precursor lacking the enzymatic activity.

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Characterization of reticulofibroblastoid colonies (CFU‐RF) derived from bone marrow and long‐term marrow culture monolayers

Lim

Izaguirre

Aye

et al. 1986

Journal Cellular Physiology

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The maintenance of hemopoietic precursors in long-term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose-clotted plasma in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU-RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid-laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU-F). The relationship of CFU-RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU-RF-derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU-RF-derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU-RF is not part of the repertoire of hemopoietic stem cells.

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New approaches to the detection of myeloperoxidase deficiency

Cited by 39 publications

References 14 publications

Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis

Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis

Genetic characterization of myeloperoxidase deficiency in Italy

Characterization of reticulofibroblastoid colonies (CFU‐RF) derived from bone marrow and long‐term marrow culture monolayers

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