RESUMO
Severe congenital neutropenia (SCN) is a genetically heterogeneous, rare disorder defined by a persistent absolute neutrophil count <500k mm(-3) with neutrophil maturation arrest at the promyelocyte stage and an increased risk for infection as well as a propensity towards developing myelodysplastic syndrome and acute myelogenous leukemia. We report a case of incidentally identified SCN in a full-term, otherwise healthy infant girl. Routine complete blood counts obtained for follow up of ABO incompatibility-induced jaundice and anemia identified mild neutropenia at birth followed by severe persistent neutropenia by 1 week of birth. Genetic testing confirmed the clinical suspicion of SCN with the identification of a mutation in the ELANE gene. Prompt identification and treatment of infants with SCN is critical to minimizing morbidity and mortality; as such, a diagnosis of SCN must be considered in all infants with neutropenia even in the absence of infection.
Assuntos
Neutropenia , Síndrome Congênita de Insuficiência da Medula Óssea , Feminino , Humanos , Recém-Nascido , Elastase de Leucócito/genética , Mutação , Neutropenia/congênito , Neutropenia/diagnóstico , Neutropenia/genéticaRESUMO
Severe congenital neutropenia (SCN) is a hematologic condition characterized by arrested maturation of myelopoiesis at the promyelocyte stage of development. With appropriate treatment using recombinant human granulocyte-colony-stimulating factor (r-HuG-CSF), SCN patients are now surviving longer, but are at increased risk of developing myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Hematopoietic stem cell transplantation (HSCT) is the only curative option for these patients, but transplantation outcomes after malignant transformation are not well established. We report results for six patients with SCN who underwent HSCT for MDS or AML between 1997 and 2001 at two transplant centers. Two patients transplanted for MDS survived. Both of these patients were transplanted without being given induction chemotherapy. Four patients, who all received induction chemotherapy for AML prior to HSCT, died. Administering induction chemotherapy prior to HSCT resulted in significant morbidity. Rapid transplantation should be the goal for the SCN patient once the diagnosis of MDS/AML is established. SCN patients should be monitored carefully for progression to MDS in order to be treated with HSCT as soon as they have progressed and before developing AML. For SCN patients who progress to AML, HSCT should still be considered, even though the risks appear to be greater.
Assuntos
Transformação Celular Neoplásica , Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia Mieloide/terapia , Neutropenia/complicações , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Exame de Medula Óssea , Criança , Pré-Escolar , Progressão da Doença , Feminino , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Lactente , Cariotipagem , Leucemia Mieloide/etiologia , Masculino , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/terapia , Neutropenia/congênito , Neutropenia/terapia , Estudos Retrospectivos , Taxa de Sobrevida , Condicionamento Pré-Transplante , Resultado do TratamentoRESUMO
Congenital neutropenia and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding neutrophil elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of neutrophil elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding neutrophil elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.
Assuntos
Elastase de Leucócito/genética , Mutação , Neutropenia/congênito , Neutropenia/enzimologia , Adolescente , Adulto , Sítios de Ligação , Células da Medula Óssea/química , Criança , Pré-Escolar , Mapeamento Cromossômico , Cromossomos Humanos Par 19 , Feminino , Humanos , Lactente , Elastase de Leucócito/química , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Estrutura Molecular , Neutropenia/genética , RNA/análise , Splicing de RNA , RNA Mensageiro/química , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Granulocyte colony-stimulating factor (G-CSF) has had a major impact on management of "severe chronic neutropenia," a collective term referring to congenital, idiopathic, or cyclic neutropenia. Almost all patients respond to G-CSF with increased neutrophils, reduced infections, and improved survival. Some responders with congenital neutropenia have developed myelodysplastic syndrome and acute myeloblastic leukemia (MDS/AML), which raises the question of the role of G-CSF in pathogenesis. The Severe Chronic Neutropenia International Registry (SCNIR), Seattle, WA, has data on 696 neutropenic patients, including 352 patients with congenital neutropenia, treated with G-CSF from 1987 to present. Treatment and patient demographic data were analyzed. The 352 congenital patients were observed for a mean of 6 years (range, 0.1-11 years) while being treated. Of these patients, 31 developed MDS/AML, for a crude rate of malignant transformation of nearly 9%. None of the 344 patients with idiopathic or cyclic neutropenia developed MDS/AML. Transformation was associated with acquired marrow cytogenetic clonal changes: 18 patients developed a partial or complete loss of chromosome 7, and 9 patients manifested abnormalities of chromosome 21 (usually trisomy 21). For each yearly treatment interval, the annual rate of MDS/AML development was less than 2%. No significant relationships between age at onset of MDS/AML and patient gender, G-CSF dose, or treatment duration were found (P >.15). In addition to the 31 patients who developed MDS/AML, the SCNIR also has data on 9 additional neutropenic patients whose bone marrow studies show cytogenetic clonal changes but the patients are without transformation to MDS/AML. Although our data does not support a cause-and-effect relationship between development of MDS/AML and G-CSF therapy or other patient demographics, we cannot exclude a direct contribution of G-CSF in the pathogenesis of MDS/AML. This issue is unclear because MDS/AML was not seen in cyclic or idiopathic neutropenia. Improved survival of congenital neutropenia patients receiving G-CSF therapy may allow time for the expression of the leukemic predisposition that characterizes the natural history of these disorders. However, other factors related to G-CSF may also be operative in the setting of congenital neutropenia. (Blood. 2000;96:429-436)
Assuntos
Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Leucemia Mieloide Aguda/etiologia , Síndromes Mielodisplásicas/etiologia , Neutropenia/congênito , Neutropenia/tratamento farmacológico , Adolescente , Adulto , Envelhecimento , Transformação Celular Neoplásica , Criança , Pré-Escolar , Feminino , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Humanos , Lactente , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologia , Neutropenia/genética , Fatores de TempoRESUMO
Polymorphonuclear leukocyte (PMNL) phagocytosis mediated by FcgammaRII proceeds in concert with activation of the mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase ERK2. We hypothesized that myosin light chain kinase (MLCK) could be phosphorylated and activated by ERK, thereby linking the MAP kinase pathway to the activation of cytoskeletal components required for pseudopod formation. To explore this potential linkage, PMNLs were challenged with antibody-coated erythrocytes (EIgG). Peak MLCK activity, 3-fold increased over controls, occurred at 4 to 6 minutes, corresponding with the peak rate of target ingestion and ERK2 activity. The MLCK inhibitor ML-7 (10 micromol/L) inhibited both phagocytosis and MLCK activity to basal values, thereby providing further support for the linkage between the functional response and the requirement for MLCK activation. The MAPK kinase (MEK) inhibitor PD098059 inhibited phagocytosis, MLCK activity, and ERK2 activity by 80% to 90%. To directly link ERK activation to MLCK activation, ERK2 was immunoprecipitated from PMNLs after EIgG ingestion. The isolated ERK2 was incubated with PMNL cytosol as a source of unactivated MLCK and with MLCK substrate; under these conditions ERK2 activated MLCK, resulting in phosphorylation of the MLCK substrate or of the myosin light chain itself. Because MLCK activates myosin, we evaluated the effect of directly inhibiting myosin adenosine triphosphatase using 2,3-butanedione monoxime (BDM) and found that phagocytosis was inhibited by more than 90% but MLCK activity remained unaffected. These results are consistent with the interpretation that MEK activates ERK, ERK2 then activates MLCK, and MLCK activates myosin. MLCK activation is a critical step in the cytoskeletal changes resulting in pseudopod formation.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Neutrófilos/imunologia , Fagocitose , Azepinas/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Eritrócitos/imunologia , Flavonoides/farmacologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Miosinas/antagonistas & inibidores , Naftalenos/farmacologia , Neutrófilos/enzimologia , Fagocitose/efeitos dos fármacos , FosforilaçãoRESUMO
Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS. It was also found that the actin cytoskeleton in WAS platelets is capable of producing the hallmark cytoarchitectural features associated with activation. Further analysis revealed accelerated cell death in WAS lymphocytes as evidenced by increased caspase-3 activity. This increased activity resulted in accelerated apoptosis of these cells. CD95 expression was also increased in these cells, suggesting an up-regulation in the FAS pathway in WAS lymphocytes. Additionally, inhibition of actin polymerization in lymphocytes using cytochalasin B did not accelerate apoptosis in these cells. This suggests that the accelerated apoptosis observed in WAS lymphocytes was not secondary to an underlying defect in actin polymerization caused by mutation of the WAS gene. These data indicate that WASP does not play a universal role in signaling actin polymerization, but does play a role in delaying cell death. Therefore, the principal consequence of mutations in the WAS gene is to accelerate lymphocyte apoptosis, potentially through up-regulation of the FAS-mediated cell death pathway. This accelerated apoptosis may ultimately give rise to the clinical manifestations observed in WAS. (Blood. 2000;95:1283-1292)
Assuntos
Actinas/sangue , Apoptose , Plaquetas/citologia , Citoesqueleto/fisiologia , Leucócitos/citologia , Síndrome de Wiskott-Aldrich/sangue , Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Sobrevivência Celular/efeitos dos fármacos , Citocalasina B/farmacologia , Dinoprosta/farmacologia , Humanos , Técnicas In Vitro , Cinética , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Linfócitos/fisiologia , Mutação de Sentido Incorreto , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Necrose , Mutação Puntual , Proteínas/genética , Valores de Referência , Acetato de Tetradecanoilforbol/farmacologia , Trombocitopenia/sangue , Trombocitopenia/genética , Trombocitopenia/patologia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich , Receptor fas/sangue , Domínios de Homologia de srcRESUMO
In the present study, we investigated the mechanism by which sphingosine and its analogues, dihydrosphingosine and phytosphingosine, inhibit polymorphonuclear leukocyte (PMN) phagocytosis of IgG-opsonized erythrocytes (EIgG) and inhibit ERK1 and ERK2 phosphorylation. We used antibodies that recognized the phosphorylated forms of ERK1 (p44) and ERK2 (p42) (extracellular signal-regulated protein kinases 1 and 2). Sphingoid bases inhibited ERK1 and ERK2 activation and phagocytosis of EIgG in a concentration-dependent manner. Incubation with glycine, N,N'-[1, 2-ethanediylbis(oxy-2, 1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-bis[ (acetylox y)methyl]ester (BAPTA,AM), an intracellular chelator of calcium, failed to block either phagocytosis or ERK1 and ERK2 phosphorylation, consistent with the absence of a role for a calcium-dependent protein kinase C (PKC) in ERK1 and ERK2 phosphorylations. Western blotting demonstrated that sphingosine inhibited the translocation of Raf-1 and PKCdelta from PMN cytosol to the plasma membrane during phagocytosis. These data are consistent with the interpretation that sphingosine regulates ERK1 and ERK2 phosphorylation through inhibition of PKCdelta, and this in turn leads to inhibition of Raf-1 translocation to the plasma membrane. Consistent with this interpretation, the sphingosine-mediated inhibition of phagocytosis, ERK2 activation, and PKCdelta translocation to the plasma membrane could be abrogated with a cell-permeable diacylglycerol analog. The increase in the diacylglycerol mass correlated with the translocation of PKCdelta and Raf-1 to the plasma membrane by 3 minutes after the initiation of phagocytosis. Additionally, the diacylglycerol analog enhanced phagocytosis by initiating activation of PKCdelta and its translocation to the plasma membrane. Because PMN generate sufficient levels of sphingosine by 30 minutes during phagocytosis of EIgG to inhibit phagocytosis, it appears that sphingosine can serve as an endogenous regulator of EIgG-mediated phagocytosis by downregulating ERK activation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas Quinases Ativadas por Mitógeno , Neutrófilos/fisiologia , Fagocitose/efeitos dos fármacos , Esfingosina/farmacologia , Transporte Biológico/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Membrana Celular/metabolismo , Diglicerídeos/farmacologia , Eritrócitos , Humanos , Imunoglobulina G , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Proteínas Opsonizantes , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptores Fc/fisiologiaAssuntos
Hematopoese/fisiologia , Neutrófilos/citologia , Animais , Apoptose , Proteínas de Transporte/fisiologia , Adesão Celular , Quimiocinas/fisiologia , Quimiotaxia de Leucócito , Grânulos Citoplasmáticos/metabolismo , Endocitose , Endotélio Vascular/citologia , Exocitose , Terapia Genética , Doença Granulomatosa Crônica/patologia , Doença Granulomatosa Crônica/terapia , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Neutrófilos/fisiologia , Fagocitose , Explosão Respiratória , Proteína 3 Associada à Membrana da VesículaRESUMO
To better understand the mechanism of leukocyte migration in complex environments, model extracellular matrices were prepared using gelatin, Hanks' solution, Bodipy-BSA (fluorescent upon proteolysis), and dihydrotetramethylrosamine or hydroethidine (fluorescent upon oxidation). Using quantitative microfluorometry, neutrophil-mediated extracellular pulses of reactive oxygen metabolites (ROMs) and pericellular proteolysis were periodically observed showing that these functions occur as quantal bursts. However, chronic granulomatous disease neutrophils, which do not produce ROMs, did not display ROM deposition. Matrices show an alternating pattern of green (proteolytic) and red (oxidative) fluorescence, indicating these functions are out of phase. Electric fields phase-matched with metabolic oscillations, which increase the amplitude of intracellular NAD(P)H oscillations, increase ROM deposition and pericellular proteolysis; this further supports the link between intracellular chemical oscillators and extracellular functions. This phase relationship may allow ROMs to inactivate protease inhibitors, followed by protease activation.
Assuntos
Quimiotaxia de Leucócito , Endopeptidases/metabolismo , Neutrófilos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Adulto , Compostos de Boro , Matriz Extracelular , Corantes Fluorescentes , Gelatina , Humanos , Técnicas In Vitro , Oscilometria , Espectrometria de Fluorescência/métodos , Fatores de TempoRESUMO
The development of recombinant-met human granulocyte-colony stimulating factor (r-metHuG-CSF) for clinical use has had a major influence on the treatment of many diseases. This impact has perhaps been greatest for treatment of severe chronic neutropenia (SCN) conditions for which there were no predictably effective treatment before the availability of these growth factors, particularly r-metHuG-CSF (Filgrastim, Amgen Inc, Thousand Oaks, CA; or Lenograstim, Rhone-Poulenc Rorer, Milan, Italy). Based on careful studies in many countries it is now known that more than 95% of these patients will respond promptly to r-metHuG-CSF treatment with normalization of the blood neutrophil levels and reduction in the occurrence of both major and minor consequences of their severe neutropenia. The availability of this treatment will undoubtedly lead to much additional research on the mechanisms governing neutrophil production and the basic mechanisms that can cause neutropenia among patients who have SCN. Among patients who have SCN those who are diagnosed to have severe congenital neutropenia (Kostmann's syndrome) or Shwachman-Diamond syndrome are at risk of developing myelodysplasia and/or acute myelogenous leukemia. The role of r-metHuG-CSF in facilitating the risk remains to be determined. Thus, it is important that long-term evaluation of the safety and efficacy of treatment of SCN and cooperation in research on these rare conditions proceed under the auspices of an international registry monitoring the clinical outcome of patients with severe congenital neutropenia.
Assuntos
Neutropenia/fisiopatologia , Neutropenia/terapia , Doença Crônica , Contraindicações , Feminino , Filgrastim , Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Humanos , Neutropenia/congênito , Neutropenia/patologia , Neutrófilos/patologia , Periodicidade , Gravidez , Proteínas Recombinantes/uso terapêuticoRESUMO
Human peripheral blood neutrophils (PMN) plated onto fibrinogen and activated with FMLP release H2O2 and lactoferrin, a specific granule component, with parallel kinetics. Although tumor necrosis factor-alpha (TNF alpha) only primes PMN in suspension, it is a potent agonist of adherent PMN. Activation of adherent PMN by FMLP (10(-7) mol/L) stimulated detectable release of TNF alpha within 45 minutes of stimulation, with maximal release (45.5 pg/10(6) cells) detected by 90 minutes. TNF alpha release paralleled the release of both lactoferrin and H2O2. To determine if TNF alpha plays a role in H2O2 and lactoferrin release, we investigated the effect of anti-TNF alpha antibodies on FMLP-stimulated activation of adherent PMN. A neutralizing rabbit anti-TNF alpha antibody inhibited both H2O2 and lactoferrin release stimulated by FMLP, whereas rabbit lgG, anti-HLA-A,B,C, anti-CD 14, and anti-interleukin-8 antibodies were without effect. The simultaneous addition of TNF alpha (1,000 U/mL) with anti-TNF alpha antibody reversed the inhibition seen with anti-TNF alpha alone. Furthermore, treatment of PMN with either actinomycin D or cylcoheximide resulted in partial (33%) inhibition of H2O2 and lactoferrin release, suggesting that protein synthesis is required for FMLP-mediated activation of adherent PMN. The addition of TNF alpha to either cycloheximide or of actinomycin D-treated PMN overcame the inhibition, indicating that the effect was specific for TNF alpha. The addition of antibodies against either the 55-or 75-kD TNF alpha receptors (referred to as p55 and p75, respectively) resulted in partial (32%) inhibition of FMLP-mediated activation of H2O2 and lactoferrin release, whereas a combination of both antibodies reduced their release to control levels. These data indicate that both p55 and p75 are involved in FMLP activation of adherent PMN. Taken together, these findings indicate that the production of TNF alpha and ligation of TNF alpha receptors are central to FMLP activation of PMN adherent to fibrinogen.
Assuntos
Fatores Quimiotáticos/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Receptores Imunológicos/fisiologia , Receptores de Peptídeos/fisiologia , Animais , Antígenos CD/fisiologia , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Lactoferrina/metabolismo , Neutrófilos/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Coelhos , Ratos , Receptores de Formil Peptídeo , Receptores Imunológicos/efeitos dos fármacos , Receptores de Peptídeos/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Proteínas Recombinantes/farmacologia , Explosão Respiratória/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Recombinant human granulocyte colony-stimulating factor (G-CSF) treatment has been shown to increase average neutrophil counts substantially in patients with childhood-onset cyclic neutropenia (or "cyclic hematopoiesis"), but not to eliminate the cyclic oscillations of neutrophil counts or those of other blood elements (monocytes, platelets, eosinophils, and reticulocytes) that are characteristic of this hematopoietic disorder. Indeed, oscillations of neutrophil counts are amplified during G-CSF treatment. We have compared the effects of recombinant granulocyte-macrophage-CSF (GM-CSF) with those of G-CSF in three patients with this disease (2 men and 1 woman, 17, 30, and 32 years of age). These patients were treated with GM-CSF (2.1 micrograms/kg/day, subcutaneously) for 6 weeks, preceded and followed by 6 to 13 weeks of detailed observation to document changes in the cyclic oscillations of blood neutrophils and other blood elements; two of the patients were subsequently treated with G-CSF (5.0 micrograms/kg/d, subcutaneously) and observed for comparable periods of time. Unlike G-CSF treatment, which increased average neutrophil counts more than 20-fold, GM-CSF increased neutrophil counts only modestly, from 1.6- to 3.9-fold, although eosinophilia of varying prominence was induced in each patient. However, at the same time, GM-CSF treatment dampened or eliminated the multilineage oscillations of circulating blood elements (neutrophils, monocytes, platelets, and/or reticulocytes) in each of the patients. In contrast, G-CSF treatment of the same patients markedly amplified the oscillations of neutrophil counts and caused the cycling of other blood elements (monocytes in particular) to become more distinct. These findings support the conclusion that the distinctive cycling of blood cell production in childhood-onset cyclic neutropenia results from abnormalities in the coordinate regulation of both GM-CSF-responsive, multipotential progenitor cells and G-CSF-responsive, lineage-restricted, neutrophil progenitors.
Assuntos
Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Neutropenia/tratamento farmacológico , Adolescente , Adulto , Idade de Início , Medula Óssea/patologia , Criança , Ensaio de Unidades Formadoras de Colônias , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Contagem de Leucócitos/efeitos dos fármacos , Masculino , Monócitos/efeitos dos fármacos , Neutropenia/sangue , Neutropenia/fisiopatologia , Neutrófilos/efeitos dos fármacos , Contagem de Plaquetas/efeitos dos fármacos , Proteínas Recombinantes/uso terapêutico , Contagem de Reticulócitos/efeitos dos fármacos , Fatores de TempoRESUMO
Congenital neutropenia (Kostmann's syndrome [KS]) is an autosomal recessive syndrome that is characterized by profound neutropenia, resulting in major clinical infections and death. Since the neutropenia and symptoms in KS improve in response to exogenous administration of granulocyte colony-stimulating factor (G-CSF), we studied bone marrow cytokine (G-CSF, granulocyte-macrophage CSF [GM-CSF], and interleukin-6) production under both basal and stimulated conditions. No differences in G-CSF, GM-CSF, or IL-6 gene expression were found in bone marrow stromal cells between normal controls and KS patients, and all three cytokines were detected by enzyme-linked immunosorbent assay (ELISA) in medium conditioned by bone marrow stromal cells from normal donors and patients with KS. Each KS patient tested had detectable, functional G-CSF in their own serum before exogenous G-CSF administration. Since G-CSF production appeared normal in KS patients, we then asked whether we could detect structural defects in the signaling portion of G-CSF receptor genes. Polymerase chain reaction (PCR) amplification of the G-CSF receptor transmembrane region alone, and of the transmembrane plus cytosolic portions of the receptor, yielded the size products predicted from the sequences of the normal G-CSF receptor. Single-strand conformational polymorphism (SSCP) analysis of G-CSF receptor PCR products demonstrated no variance in structural conformation between KS patients and normal subjects. These results demonstrate that bone marrow stromal cells in patients with KS secrete normal concentrations of functional G-CSF and suggest that the neutropenia in KS patients is caused by an inability of neutrophilic progenitor and precursor cells to respond to normal, physiologic levels of G-CSF. Such a defect, clinically responsive to pharmacologic doses of G-CSF, might be caused by defects in the post-G-CSF receptor signal transduction pathway.
Assuntos
Fator Estimulador de Colônias de Granulócitos/biossíntese , Neutropenia/congênito , Neutropenia/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/química , Sequência de Bases , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Interleukin 8 (IL-8) is a recently described cytokine that functions as a potent neutrophil chemoattractant and activator. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) and the regulation of IL-8 gene expression to specifically test the hypothesis that ROI would induce production of IL-8 mRNA and protein. In lipopolysaccharide-stimulated human whole blood, the OH radical scavenger dimethyl sulfoxide (Me2SO) dramatically inhibited (approximately 90%) IL-8 production, but had minimal effects on the production of tumor necrosis factor, interleukin 1 beta (IL-1), and IL-6. To determine whether NADPH-oxidase-generated free radicals were critical in the regulation of IL-8, studies were performed using blood from patients with chronic granulomatous disease. In both normal individuals and patients with chronic granulomatous disease, production of IL-8 could be initiated with lipopolysaccharide, phytohemagglutinin, or aggregated immune complexes, and this production could be inhibited by Me2SO (1% v/v). To examine if oxidant stress represents a ubiquitous mechanism for the induction of IL-8, experiments were performed in cultured cell lines. In the human hepatoma cell line Hep-G2, Me2SO dose-dependently inhibited tumor necrosis factor-stimulated IL-8 production, with a 74 +/- 1% reduction observed at a Me2SO concentration of 1%. Direct exposure to ROI demonstrated that H2O2 stimulated IL-8 production in a dose-dependent manner in Hep-G2 cells, A549 pulmonary type II epithelial cells, and human skin fibroblasts; this induction could be prevented by addition of catalase. The production of IL-8 appeared to be specific to an oxidant stress since exposure of the cells to heat shock or chemical stress did not induce expression of IL-8. These studies demonstrate that oxidant stress is an important regulator of IL-8 gene expression and support the hypothesis that low levels of ROI may serve to initiate IL-8 production which then serves to recruit neutrophils to sites of inflammation.
Assuntos
Antioxidantes/farmacologia , Dimetil Sulfóxido/farmacologia , Sequestradores de Radicais Livres , Regulação da Expressão Gênica/efeitos dos fármacos , Doença Granulomatosa Crônica/sangue , Peróxido de Hidrogênio/farmacologia , Interleucina-8/biossíntese , Neutrófilos/metabolismo , Oxidantes/farmacologia , Pele/metabolismo , Sequência de Bases , Northern Blotting , Carcinoma Hepatocelular , Linhagem Celular , Sobrevivência Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Radicais Livres/metabolismo , Doença Granulomatosa Crônica/genética , Humanos , Recém-Nascido , Cinética , Lipopolissacarídeos/farmacologia , Neoplasias Hepáticas , Pulmão , Masculino , Modelos Biológicos , Dados de Sequência Molecular , NADH NADPH Oxirredutases/sangue , NADPH Oxidases , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Sondas de Oligonucleotídeos , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
In this study we test the hypothesis that reactive oxygen metabolites are delivered from neutrophils to simultaneously both the cell surface and cytosol of opsonized YAC erythroleukemic target cells. Using 5' (or 6') carboxyl-2',7'-dichlorodihydrofluorescein (H2-CDCF) diacetate as starting material, we synthesized its succinimidyl ester derivative. H2-CDCF-conjugated IgG prepared from the succinimidyl ester derivative was used to opsonize targets. In vitro studies have shown that H2-CDCF becomes fluorescent upon exposure to reactive oxygen metabolites, including hydrogen peroxide. Using video intensified epifluorescence microscopy, we observed that reactive oxygen metabolites are deposited on tumor cell membranes during neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC). This deposition process is catalase sensitive. The role of reactive oxygen metabolites produced by neutrophils in triggering the oxidation of H2-CDCF is further supported by the observation that neutrophils from chronic granulomatous disease (CGD) patients did not affect target fluorescence. YAC tumor cells were also labeled with dihydrorhodamine 123 or dihydrotetramethylrosamine. The oxidized forms of these reagents were found within the cytoplasm of YAC cells. During ADCC normal neutrophils, but not neutrophils obtained from CGD patients, triggered the oxidation of dihydrorhodamine 123 and dihydrotetramethylrosamine within tumor cells. Using two-color automated epifluorescence microscopy, we could not detect temporal intermediates with fluorescence in only one compartment, i.e., either solely on the plasma membrane or in the cytoplasm. These observations suggest that reactive oxygen metabolites cross target membranes (< 12 sec. These studies show that reactive oxygen metabolites are deposited both onto and into tumor cells during ADCC, wherein both compartments could become vulnerable to oxidant-mediated damage.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Neutrófilos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Citoplasma/fisiologia , Citoplasma/ultraestrutura , Fluoresceínas/metabolismo , Doença Granulomatosa Crônica/patologia , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Microscopia de Fluorescência , Neutrófilos/patologia , Oxirredução , Oxigênio/metabolismo , Fagocitose/fisiologia , Espécies Reativas de Oxigênio/análise , Rodaminas/metabolismo , Espectrofotometria , Células Tumorais CultivadasRESUMO
Optical microscopic techniques have been utilized to study the deposition of lactoferrin, a specific granule marker, and superoxide anions into target erythrocytes during antibody-dependent phagocytosis. Previous studies from this laboratory have shown that the entry of superoxide anions into erythrocytes can be sensitively monitored with Soret band transmitted light microscopy. When neutrophils were incubated with BAPTA/AM, an intracellular Ca2+ chelator, they phagocytosed IgG-opsonized sheep red blood cells (SRBC) but did not affect the microscopically detected absorption of their Soret band. When these same erythrocytes were observed after the infusion of 20 microM ionomycin, a Ca2+ ionophore, 58% of the cell-bound SRBC targets were destroyed immediately. However, neutrophils from chronic granulomatous disease (CGD) patients were unable to affect the Soret absorption of erythrocyte targets under any conditions. These results suggest that a Ca2+ signal can participate in triggering superoxide deposition in targets. Since Ca2+ signals are known to participate in the exocytic release of granules, we tested the hypothesis that specific lactoferrin-bearing granules are delivered to targets in parallel with superoxide anions. Lactoferrin delivery to phagosomes was monitored using resonance energy transfer (r.e.t.) microscopy. SRBCs were opsonized with both unconjugated and rhodamine B isothiocyanate (RBITC)-conjugated rabbit anti-SRBC IgG. After incubation with adherent neutrophils, the samples were washed, fixed with 3.7% paraformaldehyde, then labeled with fluorescein isothiocyanate (FITC)-conjugated antilactoferrin IgG. Energy transfer between FITC and RBITC was imaged microscopically and quantitated by photon counting. Significant levels of r.e.t. between antilactoferrin and anti-SRBC labels were observed after phagocytosis, but not in the absence of acceptor fluorochromes. To control for r.e.t. specificity, neutrophil membranes were labeled with FITC-conjugated, anti-HLA IgG after internalization of rhodamine B-tagged SRBCs (RSRBCs). Although r.e.t. between lactoferrin and RSRBCs labels was observed, no r.e.t. between HLA and RSRBC labels could be found. Further studies showed that treatment of neutrophils with BAPTA inhibited r.e.t. between anti-lactoferrin and RSRBCs. However, addition of ionomycin relieved this inhibition of energy transfer. These experiments show that both lactoferrin and superoxide delivery to targets are regulated in parallel by a Ca(2+)-dependent pathway. Furthermore, by combining Soret microscopy with r.e.t. microscopy, we have shown that superoxide anions and lactoferrin are delivered to the same phagosomes. We speculate that the NADPH oxidase, which produces superoxide anions, is assembled on specific granule membranes, thus accounting for their parallel Ca(2+)-dependence, activation, and delivery.
Assuntos
Cálcio/fisiologia , Grânulos Citoplasmáticos/fisiologia , Eritrócitos/metabolismo , Lactoferrina/metabolismo , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Cálcio/análise , Cálcio/metabolismo , Células Cultivadas , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/ultraestrutura , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Eritrócitos/citologia , Fluoresceína-5-Isotiocianato , Humanos , Imunoglobulina G/imunologia , Ionomicina/farmacologia , Lactoferrina/análise , Lactoferrina/imunologia , Microscopia/métodos , NADP/fisiologia , Neutrófilos/química , Neutrófilos/ultraestrutura , Oxigênio/metabolismo , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Fagossomos/química , Fagossomos/metabolismo , RodaminasRESUMO
OBJECTIVE: The purpose of this study was to develop standards for the normal volume of the spleen in children as measured on CT scans. SUBJECTS AND METHODS: CT scans were used to measure the volume of the spleen in 48 children (30 boys and 18 girls), 1 day to 18 years old (mean and median ages were 8.2 and 7.8 years, respectively). Children who had underlying malignant tumors, infection, hematologic diseases, or other conditions that could alter splenic size were excluded. The area of the spleen on each axial section was determined by tracing its outline on the CT monitor and measuring the area of the region of interest. The area of the spleen on each section was multiplied by the slice thickness to calculate the volume of the spleen for each section. The total volume of the spleen was then determined by adding the individual volumes for each of the sections through the spleen. This method of calculating splenic volume has been validated in previous studies in adult subjects. The volume of the spleen was analyzed as a function of both body weight and age. RESULTS: The volume of the spleen correlated better with body weight than with age. The best regression model (r = .85) was a linear relationship as follows: splenic volume (cm3) = 0.7 + [4.6 x weight (kg)]. Using these data and a regression model, we generated standards for normal volume of the spleen as a function of body weight (with 95% tolerance intervals). CONCLUSION: We have developed CT standards for normal splenic volume in children. These standards can be used to objectively measure the size of the spleen in children who have clinically suspected splenomegaly.
Assuntos
Peso Corporal , Baço/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Criança , Feminino , Humanos , Masculino , Valores de Referência , Baço/anatomia & histologia , Esplenomegalia/diagnóstico por imagemRESUMO
Neutrophil specific granule deficiency (SGD) is a rare congenital disorder of unknown cause associated with an impaired inflammatory response and an absence of neutrophil secondary granules. Reduced levels of several neutrophil proteins have led to the suggestion that the defect may lie at the level of transcription, a hypothesis that is supported by abnormally low levels of lactoferrin message in the bone marrow of two SGD patients. We have examined the level of seven granule protein RNAs in one SGD patient and have compared them with reported protein levels. We have found the RNA levels for all of these genes to be reduced in proportion to the decreased levels of their respective proteins. These data further support the hypothesis that the reduced protein levels reflect a defect in transcriptional control.
Assuntos
Proteínas Sanguíneas/deficiência , Proteínas Sanguíneas/genética , Grânulos Citoplasmáticos/metabolismo , Doenças Hematológicas/genética , Neutrófilos/ultraestrutura , RNA Mensageiro/metabolismo , Northern Blotting , Células da Medula Óssea , Colagenases/genética , Defensinas , Gelatina/metabolismo , Humanos , Lactoferrina/genética , Metaloendopeptidases/genética , Peroxidase/genética , Transcobalaminas/genética , Transcrição GênicaRESUMO
Recombinant human granulocyte-colony stimulating factor (G-CSF) and recombinant human granulocyte/macrophage-colony stimulating factor (GM-CSF) stimulate neutrophil production from precursors in the marrow and enhance granulocyte functions in vitro. We studied the effects of G-CSF and GM-CSF on neutrophil superoxide production and secretion. G-CSF and GM-CSF alone stimulated neither superoxide production nor secretion, but both agents primed neutrophils for superoxide production stimulated by either N-formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin. Optimal priming occurred with G-CSF at 5.3 ng/ml for 20 minutes and for GM-CSF at 1 ng/ml for 60 minutes. Priming by GM-CSF was more readily inhibited by the tyrosine kinase inhibitor ST638 but was unaffected by staurosporine. Conversely, G-CSF priming was inhibited by staurosporine but not by ST638. Neither protein kinase C translocation nor increased protein kinase C activity, however, were observed after G-CSF/GM-CSF treatment. Priming by G-CSF and GM-CSF was sensitive to pertussis toxin, suggesting the involvement of guanine nucleotide-binding proteins (G-proteins). Neutrophils from three siblings with cyclic neutropenia were studied to observe the effects of G-CSF treatment on neutrophil function in vivo; sibling 1 and sibling 2 were treated with G-CSF for 6 months, but sibling 3 was not in the treatment group. Compared with neutrophils from normal donors, neutrophils from sibling 1 and sibling 2 were primed in vivo for superoxide release stimulated by either ionomycin or FMLP. Superoxide released by neutrophils from sibling 3 was similar to control cells.(ABSTRACT TRUNCATED AT 250 WORDS)