Louisa V. Forbes

An activating mutation in the transmembrane domain of the granulocyte colony-stimulating factor receptor in patients with acute myeloid leukemia.

To date, constitutively activating point mutations reported in hematopoietic growth factor receptors in patients with acute myeloid leukemia (AML) have been restricted to receptors with intrinsic tyrosine kinase activity such as c-kit and FLT3. We describe here a Thr617Asn mutation in the transmembrane domain of the non-tyrosine kinase receptor for granulocyte colony-stimulating factor (G-CSF) in the blast cells of two out of 555 AML patients examined. The mutant receptor conferred growth factor independence on factor-dependent Ba/F3 cells. In the absence of ligand, immunoblotting showed weak phosphorylation of JAK2, STAT3, ERKs 1 and 2 and the receptor itself, and there was approximately 70% of maximal growth in a proliferation assay. All signals were significantly enhanced in the presence of G-CSF. Retroviral transduction of mutant receptor into primary hematopoietic CD34+ cells induced G-CSF independent myeloid differentiation as assessed by the development of neutrophils and surface expression of CD11b and CD14. These results confirm the importance of the transmembrane domain for receptor function and suggest that introduction of an asparagine residue can cause sufficient stabilization of helix–helix interactions in the absence of ligand to activate downstream signaling pathways involved in directing proliferation and differentiation.

Measuring chlorine bleach in biology and medicine

BACKGROUND Chlorine bleach, or hypochlorous acid, is the most reactive two-electron oxidant produced in appreciable amounts in our bodies. Neutrophils are the main source of hypochlorous acid. These champions of the innate immune system use it to fight infection but also direct it against host tissue in inflammatory diseases. Neutrophils contain a rich supply of the enzyme myeloperoxidase. It uses hydrogen peroxide to convert chloride to hypochlorous acid. SCOPE OF REVIEW We give a critical appraisal of the best methods to measure production of hypochlorous acid by purified peroxidases and isolated neutrophils. Robust ways of detecting it inside neutrophil phagosomes where bacteria are killed are also discussed. Special attention is focused on reaction-based fluorescent probes but their visual charm is tempered by stressing their current limitations. Finally, the strengths and weaknesses of biomarker assays that capture the footprints of chlorine in various pathologies are evaluated. MAJOR CONCLUSIONS Detection of hypochlorous acid by purified peroxidases and isolated neutrophils is best achieved by measuring accumulation of taurine chloramine. Formation of hypochlorous acid inside neutrophil phagosomes can be tracked using mass spectrometric analysis of 3-chlorotyrosine and methionine sulfoxide in bacterial proteins, or detection of chlorinated fluorescein on ingestible particles. Reaction-based fluorescent probes can also be used to monitor hypochlorous acid during phagocytosis. Specific biomarkers of its formation during inflammation include 3-chlorotyrosine, chlorinated products of plasmalogens, and glutathione sulfonamide. GENERAL SIGNIFICANCE These methods should bring new insights into how chlorine bleach is produced by peroxidases, reacts within phagosomes to kill bacteria, and contributes to inflammation. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Potent Reversible Inhibition of Myeloperoxidase by Aromatic Hydroxamates

Background: Myeloperoxidase causes oxidative damage in many inflammatory diseases. Results: New substituted aromatic hydroxamates are identified as potent, selective, and reversible inhibitors of MPO. Conclusion: Binding affinities of hydroxamates to the heme pocket determine the potency of inhibition. Significance: Compounds that bind tightly to the active site of myeloperoxidase have potential as therapeutically useful inhibitors of oxidative stress. The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.

The major phosphorylation site of the NADPH oxidase component p67phox is Thr233.

Phosphorylation of p67phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC-MS suggested that Thr233 was the phosphorylated residue. Mutagenesis of Thr233 to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr233 has been identified as the major phosphorylation site of p67phox, which is situated in a proline-rich domain.

Islet cell antibodies and antibodies against glutamic acid decarboxylase in newly diagnosed adult-onset diabetes mellitus

This study aimed to determine the prevalence of antibodies against glutamic acid decarboxylase (anti-GAD) and islet cell antibodies (ICA) in relation to beta-cell function in adults newly-diagnosed with diabetes mellitus. beta-cell function was assessed in adults aged 25-70 years newly-diagnosed with diabetes mellitus (n = 84) and control subjects (n = 34) using a 1.6 MJ mixed meal test procedure. beta-cell function was evaluated by the true insulin (defined as immunoreactive insulin minus proinsulin) response to the mixed meal test. Subjects were classified on the basis of the area under the true insulin curve (normal 16830-107700 pmol min/I) and the sum of the 30 and 60 min incremental response (normal 285-3295 pmol/I). The prevalence of anti-GAD and ICA was determined using radioimmunoprecipitation and indirect immunofluorescence, respectively. Twelve (14%) of the study cohort were insulin deficient showing little or no true insulin release. Of the insulin deficient individuals, seven (58%) subjects were anti-GAD antibody positive, compared with eleven (15%) of the subjects without insulin deficiency (P < 0.001). Seven (58%) insulin deficient subjects were ICA positive, whereas only two (3%) non-insulin deficient subjects were ICA positive (P < 0.001). Eight (67%) of the insulin deficient individuals had anti-GAD or ICA, compared with twelve (17%) of those who were not insulin deficient (P < 0.001). The positive predictive values for insulin deficiency of anti-GAD and ICA were 39 and 78% respectively. The sensitivity of both antibodies for detecting insulin deficiency was 50%. The specificity for detecting insulin deficiency was 85% for anti-GAD and 97% for ICA. Positivity for both anti-GAD and ICA gave a specificity and positive predictive value for insulin deficiency of 99%, and a sensitivity of 50%. Nearly one in seven adults presenting with diabetes mellitus as a new diagnosis are insulin deficient using our criteria. Loss of beta-cell function in two thirds of individuals who are insulin deficient can be identified by anti-GAD and ICA. Early detection of these immune markers of beta-cell damage creates the potential for immune modulation to limit such damage.

Phosphorylation of p67(phox) in the neutrophil occurs in the cytosol and is independent of p47(phox)

p67phox and p47phox are phosphorylated in the course of stimulation of the NADPH oxidase in neutrophils. Isolated neutrophil cytosol can phosphorylate both of these proteins in vitro. Phosphoamino acid analysis showed that isolated membranes can tyrosine‐phosphorylate p67phox in vitro. Further experiments with anti‐phosphotyrosine antibodies did not support a role for tyrosine phosphorylation of p67phox in the cell. A phosphopeptide analysis showed that the phosphorylation of p67phox is unchanged in the absence of p47phox. These results further characterise the phosphorylation of p67phox and provide evidence that this is a cytosolic event independent of interaction with p47phox and the membrane.

Temporal Variation in Incidence of IDDM in Canterbury, New Zealand

Objective To establish the statistical significance of observed variations over the last decade in the incidence of insulin-dependent diabetes mellitus (IDDM) in the 0- to 19-yr-old age-group and to determine whether incidence has increased in Canterbury, New Zealand. Research Design and Methods The Canterbury, New Zealand, Diabetes Registry has recorded all incidence cases of diabetes mellitus prospectively since 1982. All IDDM subjects aged 0–19 yr at diagnosis and using insulin are included in the study. Ascertainment is believed to be 100%. Prevalence was recorded at 1 January 1982 and 1 January 1990. Annual incidence for 1982–1990 was determined using age and sex cross-sectional census population denominators. The statistical significance of temporal, age, sex, and seasonal variations in incidence rates was ascertained by Poisson regression models (GLIM statistical software). Results Prevalence on 1 January 1990 was 115/100,000. Incidence rates during the 9 yr were periodic, with two major peaks—one in the early 1980s, the other in 1989 continuing into 1990. The temporal variation (P < 0.02) was not age or sex specific. Incidence rates for boys were three- to fourfold higher during peak versus trough years, with a peak level of 20.7/100,000 in 1990. For girls, there was less variation, with a peak rate of 21.6/100,000 in 1990. There has been no significant increase in IDDM incidence over time. The mean rate of incidence across all age-groups for 1982–1990 was 12.7/100,000 person-yr. A significant seasonal association to the onset of IDDM was found only in boys, with incidence rates being significantly higher in winter than in summer (P < 0.01). Conclusions IDDM in Canterbury, New Zealand, presents in cycles of incidence peaks and troughs, each spanning 2–3 yr.

Isoniazid as a substrate and inhibitor of myeloperoxidase: identification of amine adducts and the influence of superoxide dismutase on their formation.

Neutrophils ingest Mycobacteria tuberculosis (Mtb) in the lungs of infected individuals. During phagocytosis they use myeloperoxidase (MPO) to catalyze production of hypochlorous acid (HOCl), their most potent antimicrobial agent. Isoniazid (INH), the foremost antibiotic in the treatment of tuberculosis, is oxidized by MPO. It rapidly reduced compound I of MPO [k = (1.22 ± 0.05) × 10(6) M(-1) s(-1)] but reacted less favorably with compound II [(9.8 ± 0.6) × 10(2) M(-1) s(-1)]. Oxidation of INH by MPO and hydrogen peroxide was unaffected by chloride, the physiological substrate for compound I, and the enzyme was partially converted to compound III. This indicates that INH is oxidized outside the classical peroxidation cycle. In combination with superoxide dismutase (SOD), MPO oxidized INH without exogenous hydrogen peroxide. SOD must favor reduction of oxygen by the INH radical to give superoxide and ultimately hydrogen peroxide. In both oxidation systems, an adduct with methionine was formed and it was a major product with MPO and SOD. We show that it is a conjugate of an acyldiimide with amines. INH substantially inhibited HOCl production by MPO and neutrophils below pharmacological concentrations. The reversible inhibition is explained by diversion of MPO to its ferrous and compound III forms during oxidation of INH. MPO, along with SOD released by Mtb, will oxidize INH at sites of infection and their interactions are likely to limit the efficacy of the drug, promote adverse drug reactions via formation of protein adducts, and impair a major bacterial killing mechanism of neutrophils.

HLA-DQβ Typing and Non-Asp57 Alleles in IDDM and Nondiabetic Subjects in New Zealand

OBJECTIVE To determine the frequency of IDDM risk-associated HLA-DQβ alleles in New Zealanders with IDDM and nondiabetic control subjects, and to examine these as susceptibility markers in relation to IDDM incidence. RESEARCH DESIGN AND METHODS HLA-DQβ typing was conducted in 55 juvenile-onset IDDM subjects diagnosed between 1990 and 1992, and 53 nondiabetic control subjects. Allele typing was conducted by a polymerase chain reaction-restriction fragment-length polymorphism technique. All subjects were residents of Canterbury, New Zealand. IDDM incidence data were obtained from the Canterbury, New Zealand, Diabetes Registry. RESULTS The frequency of the susceptibility genotype DQβ*0201/0302 was 43.6 and 5.7% in the IDDM and control groups, respectively, reflecting the increased prevalence of allele 0302 in the IDDM group. Alleles 0301, 0501, and 0602.3 were more prevalent in the control group than the IDDM group. The frequency of non-Asp57 alleles was 90.9 and 61.3% in the IDDM and control groups, respectively. Overall, the HLA-DQβ allele distribution was similar to reports from other Caucasian populations. The 0- to 19-yr age-specific IDDM incidence rate over the period in which the diabetic subjects were diagnosed was 19.5/100,000 person-yr, the highest levels observed in Canterbury over the last decade. Our relatively high background prevalence of non-Asp57 alleles and high IDDM incidence rates were similar to results from some Scandinavian and other hotspot populations. CONCLUSIONS HLA-DQβ alleles are genetic susceptibility markers in New Zealand, and other Caucasian populations. Peak IDDM incidence levels observed in 1990–1992 in our population are in accordance with a high background population prevalence of non-Asp57 alleles. These results suggest that the high Canterbury incidence rates may be due to a large HLA-DQβ non-Asp57 at-risk population.

Immunogenetic, Clinical, and Demographic Characterization of Childhood Type I Diabetes in New Zealand

OBJECTIVE To examine the relationship between genetic susceptibility alleles and islet cell antibodies (ICAs) in type I diabetes. RESEARCH DESIGN AND METHODS The human leukocyte antigen (HLA)-DQB1 alleles and ICA levels of all incident type I diabetic cases in patients < 20 years of age diagnosed in 1990 and 1991 in Canterbury, New Zealand, were determined. RESULTS The mean annual incidence rate for type I diabetes over the 24 months was 19.0/100,000 patient-years (95% confidence interval [CI] 13.5−26.0/100,000), which was considerably higher than rates observed between 1982 and 1989 (11.7/100,000; 95% CI 9.6−14.3/100,000). ICAs ≥ 10 Juvenile Diabetes Foundation units (JDF U) were present in 84.6% of the subjects, but there was a higher prevalence of ICA-negative (ICA−) subjects among those diagnosed during the winter months. The frequencies of the susceptibility allele DQB1*0302 and susceptibility genotype 0302/0201 were 71.8% and 43.5%, respectively. Subjects with 0302 tended to be younger (mean age 8.3 ± 5.1 years) than those with nonsusceptibility types (mean age 11.8 ± 4.7 years, P = 0.056). The probability of being ICA positive (ICA+) was not significantly different between subjects with allele 0302 (85.7%) and those without it (81.8%). All seven patients negative for ICA were homozygous for DQB1 nonaspartate-57. There was no clustering of the immunogenetic markers with demographic and clinical characteristics apart from age at diagnosis. CONCLUSIONS No direct relationship was observed between DQB1-defined genetic susceptibility and ICA at diagnosis, suggesting that variations at the DQB1 locus are not linked to the expression of this autoimmune marker of β-cell destruction.