RESUMO
Chronic granulomatous disease (CGD) should be considered as a differential diagnosis in children and adolescents with frequent infections, especially when caused by certain specific pathogens.This case report describes a 64-year-old female with multiple recurrent and complicated bronchopulmonary infections, caused by common, but also rare pathogens, autoimmune phenomena, malignancies and recurrent organizing pneumonia (OP) with granulomas. Finally, the patient was diagnosed with p47phox-deficient chronic granulomatous disease (CGD).Individuals with a primary immunodeficiency may survive multiple complications and may be diagnosed at an advanced age especially if the affected structure shows residual activity. When confronted with patients with recurrent bronchopulmonary infections, especially with certain specific rare pathogens, in combination with organizing pulmonary granulomas as well as autoimmune phenomena, CGD should be considered even in elderly patients. Delayed diagnosis significantly increases mortality and morbidity in such cases.
Assuntos
Doença Granulomatosa Crônica/diagnóstico , Pneumonia/diagnóstico , Diagnóstico Diferencial , Feminino , Doença Granulomatosa Crônica/complicações , Humanos , Infecções , Pessoa de Meia-Idade , Pneumonia/etiologiaRESUMO
INTRODUCTION: Complement immunodeficiencies (excluding hereditary angioedema and mannose binding lectin deficiency) are rare. Published literature consists largely of case reports and small series. We collated data from 18 cities across Europe to provide an overview of primarily homozygous, rather than partial genotypes and their impact and management. METHODS: Patients were recruited through the ESID registry. Clinical and laboratory information was collected onto standardized forms and analyzed using SPSS software. RESULTS: Seventy-seven patients aged 1 to 68 years were identified. 44 % presented in their first decade of life. 29 % had C2 deficiency, defects in 11 other complement factors were found. 50 (65 %) had serious invasive infections. 61 % of Neisseria meningitidis infections occurred in patients with terminal pathway defects, while 74 % of Streptococcus pneumoniae infections occurred in patients with classical pathway defects (p < 0.001). Physicians in the UK were more likely to prescribe antibiotic prophylaxis than colleagues on the Continent for patients with classical pathway defects. After diagnosis, 16 % of patients suffered serious bacterial infections. Age of the patient and use of prophylactic antibiotics were not associated with subsequent infection risk. Inflammatory/autoimmune diseases were not seen in patients with terminal pathway, but in one third of patients classical and alternative pathway defects. CONCLUSION: The clinical phenotypes of specific complement immunodeficiencies vary considerably both in terms of the predominant bacterial pathogen, and the risk and type of auto-inflammatory disease. Appreciation of these phenotypic differences should help both immunologists and other specialists in their diagnosis and management of these rare and complex patients.
Assuntos
Proteínas do Sistema Complemento/deficiência , Proteínas do Sistema Complemento/genética , Síndromes de Imunodeficiência/epidemiologia , Síndromes de Imunodeficiência/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Consanguinidade , Bases de Dados Factuais , Gerenciamento Clínico , Europa (Continente)/epidemiologia , Feminino , Genótipo , Humanos , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/terapia , Lactente , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
In 2009, a federally funded clinical and research consortium (PID-NET, http://www.pid-net.org) established the first national registry for primary immunodeficiencies (PID) in Germany. The registry contains clinical and genetic information on PID patients and is set up within the framework of the existing European Database for Primary Immunodeficiencies, run by the European Society for Primary Immunodeficiencies. Following the example of other national registries, a central data entry clerk has been employed to support data entry at the participating centres. Regulations for ethics approvals have presented a major challenge for participation of individual centres and have led to a delay in data entry in some cases. Data on 630 patients, entered into the European registry between 2004 and 2009, were incorporated into the national registry. From April 2009 to March 2012, the number of contributing centres increased from seven to 21 and 738 additional patients were reported, leading to a total number of 1368 patients, of whom 1232 were alive. The age distribution of living patients differs significantly by gender, with twice as many males than females among children, but 15% more women than men in the age group 30 years and older. The diagnostic delay between onset of symptoms and diagnosis has decreased for some PID over the past 20 years, but remains particularly high at a median of 4 years in common variable immunodeficiency (CVID), the most prevalent PID.
Assuntos
Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/epidemiologia , Sistema de Registros , Adolescente , Adulto , Fatores Etários , Idoso , Criança , Pré-Escolar , Bases de Dados Factuais , Feminino , Alemanha , Humanos , Síndromes de Imunodeficiência/genética , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Prevalência , Adulto JovemRESUMO
Diabetes mellitus is an important risk factor for cardiovascular diseases. Clinical evidence supports a link between hyperglycemia, endothelial dysfunction, and vascular disorders. However, the precise molecular mechanisms causing endothelial dysfunction in diabetic patients remain unclear. An interesting novel mediator could be chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), which plays an essential role in glucose metabolism. COUP-TFII is known to be expressed in venous endothelial cells. In this study, we show COUP-TFII expression in human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells. HUVECs express glucose transporters 1, 3, 6, and 10, and the insulin receptor. Insulin in combination with glucose activates protein kinase B (PKB or Akt) phosphorylation via phosphoinositide 3-kinase (PI3-kinase). Short-term (60-240 min) stimulation of HUVECs with high glucose increased COUP-TFII expression independent of insulin. Long-term (48 h) stimulation of HUVECs with high glucose augmented expression of the insulin receptor and E-selectin, but downregulated COUP-TFII protein expression. Downregulation of COUP-TFII by shRNA leads to downregulation of E-selectin and upregulation of eNOS and glucose transporters. Our data suggest that COUP-TFII is regulated by glucose in a time- and dose-dependent manner in endothelial cells. COUP-TFII might affect endothelial function in a diabetic background.
Assuntos
Fator II de Transcrição COUP/metabolismo , Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Western Blotting , Fator II de Transcrição COUP/antagonistas & inibidores , Fator II de Transcrição COUP/genética , Células Cultivadas , Vasos Coronários/citologia , Relação Dose-Resposta a Droga , Selectina E/genética , Selectina E/metabolismo , Endotélio Vascular/metabolismo , Humanos , Insulina/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Veias Umbilicais/citologiaRESUMO
We report on a 22-year-old girl with a history of recurrent febrile episodes, chronic arthritis, urticarial rash, and neurological symptoms including right hemiparesis, internal hydrocephalus, mental retardation, progressive deafness, and visual impairment. Treatment starting at age 20 months, including different combinations of immunosuppressive and antiinflammatory drugs such as corticosteroids and anti-TNFalpha antibody, was unsuccessful. Four years ago, we found a heterozygous S595G mutation in the NLRP3 gene of this patient. This prompted us to introduce anakinra, which resulted in considerable improvement of the patient's complaints.
Assuntos
Alelos , Proteínas de Transporte/genética , Síndromes Periódicas Associadas à Criopirina/genética , Análise Mutacional de DNA , Triagem de Portadores Genéticos , Adolescente , Antirreumáticos/uso terapêutico , Criança , Pré-Escolar , Síndromes Periódicas Associadas à Criopirina/diagnóstico , Síndromes Periódicas Associadas à Criopirina/tratamento farmacológico , Síndromes Periódicas Associadas à Criopirina/imunologia , Feminino , Seguimentos , Humanos , Lactente , Proteína Antagonista do Receptor de Interleucina 1/deficiência , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/sangue , Proteína 3 que Contém Domínio de Pirina da Família NLR , Adulto JovemRESUMO
Chronic infantile neurological cutaneous and articular (CINCA) syndrome is an autoinflammatory disease, defined by the triad of urticarial rash, neurological manifestations, and arthropathy, accompanied by recurrent fevers and systemic inflammation. Increasing neurological deficits result from aseptic meningitis. Sensorineural hearing loss and progressive loss of vision caused by keratoconjunctivitis or papilloedema may emerge. An autosomal-dominant inheritance is suspected although sporadic cases are reported frequently. Sixty per cent of CINCA patients carry mutations in the cold-induced autoinflammatory syndrome (CIAS1) gene. We report the favourable response of a 23-year-old CINCA patient without CIAS1 mutations to treatment with the recombinant interleukin-1 (IL-1) receptor antagonist anakinra.
Assuntos
Proteínas de Transporte/genética , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/antagonistas & inibidores , Mutação/genética , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças Reumáticas/tratamento farmacológico , Urticária/tratamento farmacológico , Adulto , Antirreumáticos/uso terapêutico , Feminino , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Doenças do Sistema Nervoso/genética , Doenças Reumáticas/genética , Síndrome , Urticária/genéticaRESUMO
Granulocytes and monocytes/macrophages from patients suffering from chronic granulomatous disease (CGD) are ineffective in killing specific kinds of phagocytized bacteria, e.g., Staphylococcus aureus, due to decreased or lacking ability to produce reactive oxygen intermediates. Commonly used antibiotics like flucloxacillin are of limited therapeutic value, because the staphylococci are protected against their action in the interior of phagocytes. However, encapsulation of flucloxacillin into liposomes could enable its entrance into the interior of neutrophils from two CGD patients to kill phagocytized bacteria there. The effect of rifampicin against intracellular staphylococci could be similarly enhanced by liposome encapsulation. Dose-response relations and kinetics of killing of intracellular bacteria by antibiotics in the free and encapsulated form were studied under different conditions using J 774 mouse macrophages, because phagocytes from CGD patients are not available in great amounts. Preincubation of phagocytes with either antibiotic in liposomes subsequently endowed the cells with a strongly enhanced ability to kill phagocytized bacteria. Our data show that a drug which normally will not reach a phagosome can be delivered to this intracellular compartment by a liposome. A possible clinical use is discussed.
Assuntos
Antibacterianos/administração & dosagem , Doença Granulomatosa Crônica/imunologia , Fagócitos/imunologia , Staphylococcus/efeitos dos fármacos , Portadores de Fármacos , Floxacilina/administração & dosagem , Humanos , Lipossomos/administração & dosagem , Neutrófilos/imunologia , Fagocitose , Rifampina/administração & dosagemRESUMO
The predominant genetic defect causing p47-phox-deficient chronic granulomatous disease (A47 degrees CGD) is a GT deletion (DeltaGT) at the beginning of exon 2. No explanation exists to account for the high incidence of this single mutation causing a rare disease in an unrelated, racially diverse population. In each of 34 consecutive unrelated normal individuals, both the normal and mutant DeltaGT sequences were present in genomic DNA, suggesting that a p47-phox related sequence carrying DeltaGT exists in the normal population. Screening of genomic bacteriophage and YAC libraries identified 13 p47-phox bacteriophage and 19 YAC clones. The GT deletion was found in 11 bacteriophage and 15 YAC clones. Only 5 exonic and 33 intronic differences distinguished all DeltaGT clones from all wild-type clones. The most striking differences were a 30-bp deletion in intron 1 and a 20-bp duplication in intron 2. These results provide good evidence for the existence of at least one highly homologous p47-phox pseudogene containing the DeltaGT mutation. The p47-phox gene and pseudogene(s) colocalize to chromosome 7q11.23. This close linkage, together with the presence within each gene of multiple recombination hot spots, suggests that the predominance of the DeltaGT mutation in A47 degrees CGD is caused by recombination events between the wild-type gene and the pseudogene(s).
Assuntos
Doença Granulomatosa Crônica/genética , Mutação , Fosfoproteínas/genética , Pseudogenes , Bacteriófagos/genética , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura/genética , Cromossomos Humanos Par 7 , Éxons , Dosagem de Genes , Variação Genética , Biblioteca Genômica , Doença Granulomatosa Crônica/etiologia , Humanos , Íntrons , NADPH Desidrogenase , NADPH Oxidases , Neutrófilos/enzimologia , Fosfoproteínas/deficiência , Reação em Cadeia da Polimerase , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
BACKGROUND: Chronic granulomatous disease (CGD) is caused by mutations in genes encoding nicotinamide dinucleotide phosphate (NADPH) oxidase subunits. CASE REPORT: A boy was diagnosed as having juvenile sarcoidosis because he presented with cervical and pulmonary lymphadenopathy with epitheloid cells and granuloma formation and high angiotensin converting enzyme. Later, a liver abscess was diagnosed. CGD was established by a dihydrorhodamine 123 (DHR) assay and genetic analysis revealed an unusual intra-exonic splice mutation in the CYBB gene encoding gp91-phox. It did not change the amino acid sequence and allowed for residual NADPH oxidase activity explaining the late onset of the disease. CONCLUSION: CGD is an important differential diagnosis of juvenile sarcoidosis.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/genética , Glicoproteínas de Membrana/genética , NADPH Oxidases/genética , Sarcoidose/diagnóstico , Criança , Diagnóstico Diferencial , Éxons/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Masculino , Mutação/genética , NADP/genética , NADPH Oxidase 2RESUMO
G-CSF and GM-CSF are hematopoietic growth factors required for proliferation and differentiation of hematopoietic precursors. G-CSF is now widely used to overcome neutropenias of various origins. Beside the absolute number, the functional capacity of neutrophils at sites of inflammation is of major importance in host defense. This review summarizes major functional and phenotypical features of neutrophils induced by G-CSF treatment in patients with acquired and congenital neutropenias. Furthermore, we focus on the differential effect of G-CSF and GM-CSF on neutrophil function in vitro and in vivo. Some of the altered abilities of cytokine-induced neutrophils are important to understand side-effects of G-CSF therapy.
Assuntos
Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos adversos , Neutropenia/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Adesão Celular , Células Cultivadas/efeitos dos fármacos , Quimiotaxia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Ativação de Neutrófilo , Neutrófilos/fisiologia , Fagocitose , Síndrome de Sweet/induzido quimicamente , Vasculite Leucocitoclástica Cutânea/induzido quimicamenteRESUMO
We tested several of the functions of macrophages (M phi) in the early phase after allogeneic bone marrow transfer to get information about this important aspect of the nonspecific immune system in the T-cell-deficient recipient. On days 3-5 after transfer, the number of M phi was reduced in the spleen, liver, lungs, and peritoneal cavity (Pe). The phagocytosis of sheep red blood cells (SRBC) by these M phi was normal or even enhanced, as in the case of Pe-M phi. Already on days 8-12 after transfer, the number of M phi in spleen and liver exceeded that of controls, whereas the number was still reduced in lungs and Pe. We examined their ability to kill P815 tumor cells, to produce tumor necrosis factor-alpha (TNF alpha), to phagocytose SRBC, to produce reactive oxygen intermediates (ROI) in vitro and to kill Listeria monocytogenes in vivo. Most functions were normal and often even enhanced, depending on the organ origin, but the ability of Pe-M phi to produce ROI was reduced. Proliferative response to macrophage colony-stimulating factor (M-CSF) and killing of YAC-1 tumor cells revealed a high frequency of macrophage precursor cells in the spleen and liver and a high natural killer (NK) activity in the liver. Altogether, enhanced nonspecific immune function, especially preactivated M phi, may enable chimeras to survive attacks by opportunistic pathogens.
Assuntos
Transplante de Medula Óssea , Macrófagos/fisiologia , Animais , Medula Óssea/imunologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Contagem de Colônia Microbiana , Fatores Estimuladores de Colônias/farmacologia , Listeria monocytogenes , Fígado/citologia , Fígado/microbiologia , Fígado/fisiologia , Pulmão/citologia , Macrófagos/imunologia , Macrófagos/transplante , Masculino , Camundongos , Camundongos Endogâmicos , Oxigênio/metabolismo , Cavidade Peritoneal/citologia , Cavidade Peritoneal/metabolismo , Quimera por Radiação , Baço/citologia , Baço/microbiologia , Baço/fisiologia , Transplante Homólogo , Células Tumorais Cultivadas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The visible excitation and emission wave-lengths of the recently developed fluorescent Ca2+ indicator fluo-3 permit analysis of the intracellular Ca2+ concentration, [Ca2+]i, in flow cytometry with a 488-nm argon laser. The role of [Ca2+]i in human polymorphonuclear leukocyte heterogeneity was investigated in response to formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and interleukin 8/neutrophil attractant/activation protein 1 (IL-8/NAP-1) by flow cytometry. The [Ca2+]i changes in different subpopulations within a heterogeneous cell suspension were resolved upon stimulation with fMLP. Using an anti-CD16 phycoerythrin-conjugated antibody and fluo-3 simultaneously, neutrophils affected and nonaffected in Ca2+ mobilization were distinguished in two patients suffering from glycogen storage disease type 1b. In normal neutrophils, a different time course of Ca2+ mobilization of neutrophil subpopulations immediately after stimulation with fMLP was detected. In addition, after stimulation with a low concentration of IL-8/NAP-1 (10(-10) M) two subsets of neutrophils appeared; one of them showed an increase in [Ca2+]i, while the other did not. These results indicate heterogeneity in the neutrophil signal transduction process involved in Ca2+ mobilization. Therefore, flow cytometric analyses can resolve changes in single-cell [Ca2+]i distribution patterns, which is important for the understanding of [Ca2+]i in neutrophil heterogeneous activation processes.
Assuntos
Cálcio/metabolismo , Complemento C5a/farmacologia , Citoplasma/metabolismo , Interleucina-8/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Compostos de Anilina , Citoplasma/efeitos dos fármacos , Citometria de Fluxo , Humanos , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Espectrometria de Fluorescência , Fatores de Tempo , XantenosRESUMO
Severe congenital neutropenia (SCN) can be corrected in vivo by treatment with pharmacological dosages of recombinant human granulocyte colony-stimulating factor (rhG-CSF). In order to analyze the decreased chemotaxis of neutrophils from SCN patients receiving rhG-CSF, neutrophil functions essential for chemotaxis were investigated. The mobilization of cytosolic calcium ([Ca2+]i) and the functional state of cytoskeletal proteins in neutrophils from SCN patients were compared with either neutrophils from healthy donors (or, in selected experiments, from patients with cyclic neutropenia) and neutrophils from patients with chemotherapy-induced neutropenia also receiving rhG-CSF. Using flow cytometric analysis, two neutrophil subpopulations were detected in SCN patients in response to N-formylmethionine leucyl-phenylalanine (FMLP) (10(-9) M to 10(-7) M), one of which was unable to respond to this stimulus with an increase in [Ca2+]i. Whereas a homogeneous increase in [Ca2+]i in normal neutrophils occurred at 10(-9) M FMLP, neutrophils from SCN patients required 10(-6) M FMLP to respond homogeneously with an increase in [Ca2+]i. In contrast, G-CSF induced neutrophils from patients with cyclic neutropenia and from patients with chemotherapy-induced neutropenia showed a normal increase in [Ca2+]i after stimulation. The [Ca2+]i-dependent superoxide anion (O2-) generation in response to FMLP was also significantly diminished in neutrophils from SCN patients compared to normal neutrophils. However, O2- generation elicited by phorbolester (PMA), which directly activates protein kinase C (PKC), was not affected in SCN neutrophils. The total immunoreactive actin content and basal F-actin content in neutrophils from SCN patients were elevated as compared to normal neutrophils and neutrophils from patients with chemotherapy-induced neutropenia. The increase in F-actin content following FMLP activation was much lower in neutrophils from SCN patients as compared with normal neutrophils. These data suggest a defect in the signal transduction pathway in neutrophils from SCN patients between FMLP ligand-receptor interaction and Ca2+ mobilization, whereas upstream of PKC, triggered events seem to be unaffected. Therefore, [Ca2+]i-dependent neutrophil function in response to FMLP, such as actin disassembly, chemotaxis and O2- generation are diminished in SCN neutrophils. The pathomechanism responsible for the defective [Ca2+]i increase might be an initial step in understanding the underlying pathophysiology of SCN.
Assuntos
Cálcio/sangue , Neutropenia/congênito , Neutrófilos/fisiologia , Transdução de Sinais/fisiologia , Actinas/sangue , Ânions , Quimiotaxia de Leucócito , Citosol/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutropenia/sangue , Neutropenia/induzido quimicamente , Superóxidos/sangue , Tubulina (Proteína)/sangueRESUMO
Apoptosis via CD95 and its ligand is an important mechanism that prevents uncontrolled proliferation of activated lymphocytes and regulates lymphocyte homeostasis. The apoptosis receptor CD95 is a transmembrane protein with an intracellular domain well conserved between CD95 and tumor necrosis factor receptor I, another apoptosis-inducing protein. Because of its functional importance, this domain was designated the death domain. We describe the molecular analysis of the CD95 death domain in a family with autoimmune lymphoproliferative syndrome (Canale-Smith syndrome), T-cell lymphoma, and Hodgkin's disease. A functional defect in apoptosis was detected in cells from the index patient, a 5-year-old girl suffering from Canale-Smith syndrome and a T-cell lymphoma, as well as in her father, who had a history of splenomegaly and mild hemolysis, and her paternal uncle who had been cured of Hodgkin's disease (HD). Expansion of double-negative T cells (CD4-CD8-) was only seen in the index patient. All family members with a functional defect in apoptosis were heterozygous for a point mutation in the death domain of CD95 (A1009G, E256G). We conclude that, within the same family, a defect in apoptosis due to a mutation in the CD95 death domain can be associated with diverse clinical phenotypes, including mild, reversible symptoms and different malignancies.
Assuntos
Apoptose/genética , Doenças Autoimunes/patologia , Doença de Hodgkin/patologia , Linfoma de Células T/patologia , Transtornos Linfoproliferativos/patologia , Receptor fas/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Sequência de Bases , Pré-Escolar , Primers do DNA , Feminino , Doença de Hodgkin/genética , Doença de Hodgkin/imunologia , Humanos , Imunofenotipagem , Linfoma de Células T/genética , Linfoma de Células T/imunologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/imunologia , Masculino , Linhagem , Mutação PuntualRESUMO
The monoclonal antibody (mAb) 31D8 has previously been described to bind more avidly to functionally active neutrophils and proved to be useful as a differentiation marker of neutrophils. However, attempts to further characterize the antigen recognized by 31D8 have not been successful. Studying the altered Fcgamma-receptor expression of human neutrophils induced by granulocyte colony-stimulating factor (G-CSF) in vivo, we could demonstrate a parallel decrease in the expression of 31D8 and the CD16 antigen. Furthermore, 31D8 showed a binding pattern on leukocyte subsets similar to that of clustered CD16 antibodies, exhibiting identical cells in double-staining experiments. Preincubation of neutrophils with 31D8 resulted in a dose-dependent inhibition of the binding of immune complexes. A decreased expression of the 31D8 antigen was found on the same cell clones to the same extent as found for the 3G8 antigen on neutrophils from patient with paroxysmal nocturnal hemoglobinuria (PNH). Treatment of polymorphonuclear leukocytes (PMN) with PIPLC resulted in a dose-dependent decrease of mAb 31D8 binding, showing that the 31D8 antigen is phosphatidylinositolglycan (PIG)-anchored. Moreover, 31D8 competed with the binding of antibodies (such as mAb 3G8) directed against the binding site of FcgammaRIII for the Fc-part of IgG. However, this mAb did not influence the binding of a CD16 antibody (mAb B73.1) which recognizes an epitope elsewhere on the CD16 antigen. We conclude from our experiments that the mAb 31D8 binds with high avidity to fcgammaRIII (CD16 antigen). Furthermore, our data indicate that its binding site is most probably located at the binding site for the Fc-part of IgG.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Receptores de IgG/imunologia , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/metabolismo , Ligação Competitiva , Células Cultivadas , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Hemoglobinúria Paroxística/imunologia , Humanos , Células Matadoras Naturais/imunologia , Leucócitos/imunologia , Neutrófilos/imunologiaRESUMO
We describe the history of a girl with interferon-gamma-receptor (IFNgammaR1) deficiency and studies performed to identify the molecular and clinical characteristics of this recently discovered disorder. This is the first report of a child from Northern Europe with IFNgammaR1 deficiency. The patient, now 7 years old, first presented with swelling and reddening at the Bacille Calmette-Guerin (BCG) vaccination site, swelling of lymph nodes, hepatomegaly, and an unusually severe varicella rash at the age of 4 months. At that time, she was diagnosed with BCG histiocytosis without typical granuloma formation and was treated with antituberculous agents. During the clinical course of her illness, several different types of atypical mycobacteria and (for the first time in an IFNgammaR1-deficient patient) Listeria monocytogenes were detected. Flow cytometric analysis showed that the patient's monocytes could not bind a monoclonal antibody specific for the IFNgamma-receptor. Our analysis of mRNA derived from the alpha-chain (IFNgammaR1) gene of this receptor revealed deletions of 173 bp and 4 bp in cDNA sequences originating from individual alleles. The 173 bp deletion was located between nucleotide positions 200 and 372, exactly matching those of exon 3, and the 4 bp deletion was located between nucleotide positions 561 and 564 of the coding region of the cDNA. Analysis of genomic DNA revealed the presence of a G to T transition at the 5'end of the splice consensus sequence of intron 3, which explains the absence of exon 3. The other allele carried the 4-base-pair deletion (ACTC) at nucleotide positions 15-18 of exon 5. Twelve months after an allo\geneic bone marrow transplantation, the patient had clinically improved.
Assuntos
Transplante de Medula Óssea , Listeriose/etiologia , Infecções por Mycobacterium não Tuberculosas/etiologia , Infecção por Mycobacterium avium-intracellulare/etiologia , Receptores de Interferon/deficiência , Alelos , Vacina BCG/efeitos adversos , Criança , Análise Mutacional de DNA , DNA Complementar/genética , Éxons/genética , Feminino , Predisposição Genética para Doença , Histiocitose/etiologia , Humanos , Interferon gama/farmacologia , Cirrose Hepática/etiologia , Linfadenite/etiologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Linhagem , Receptores de Interferon/genética , Recidiva , Deleção de Sequência , Fator de Necrose Tumoral alfa/metabolismo , Receptor de Interferon gamaRESUMO
Chronic granulomatous disease is an inherited disease characterized by the inability of phagocytes to generate normal amounts of superoxide, leaving patients susceptible to opportunistic, life-threatening infections. In the majority of cases, cytochrome b558 is absent in the X-chromosomal form of CGD. However, the neutrophils from six of nine X-linked CGD patients, reported here, expressed normal or decreased amounts of this cytochrome and are referred to as "variant" forms. In three of these six variant patients, a roughly proportional decrease in cytochrome b558 expression and production of H2O2 were found. In two cases this phenotype could be well explained by special splice mutations, whereas in the third case it was caused by a missense mutation, predicting Ser 193-->Phe. In the other three variant patients, cytochrome b558 expression and H2O2 production were clearly disproportionate as the generation of H2O2 was much more decreased than cytochrome expression. Missense mutations also were found in these cases. One of these mutations, predicting Leu 546-->Pro and affecting the putative nicotinamide adenine dinucleotide phosphate binding site, led to normal levels of cytochrome b558 expression and reduced H2O2 production. In the other two mutations, predicting Pro 339-->His and His 338-->Tyr, the putative flavin adenine dinucleotide binding site was affected. This could explain the corresponding uncommon phenotypes, characterized by zero or trace amounts of H2O2 production and the expression of relatively high amounts of nonfunctional or low functional cytochrome b558, respectively. The only missense mutation found that prevented the expression of any cytochrome b558 was caused by a predicted His 222-->Arg exchange in one of the three classic cases. The two other classic phenotypes were caused by splice mutations.
Assuntos
Grupo dos Citocromos b/genética , Heterogeneidade Genética , Doença Granulomatosa Crônica/genética , Glicoproteínas de Membrana/genética , NADPH Oxidases/genética , Mutação Puntual , Cromossomo X/genética , Adolescente , Adulto , Substituição de Aminoácidos , Sítios de Ligação , Criança , Pré-Escolar , Grupo dos Citocromos b/deficiência , Mecanismo Genético de Compensação de Dose , Feminino , Frequência do Gene , Genótipo , Alemanha , Humanos , Peróxido de Hidrogênio/metabolismo , Substâncias Macromoleculares , Masculino , Glicoproteínas de Membrana/deficiência , Pessoa de Meia-Idade , NADP/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/deficiência , Fenótipo , Splicing de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Espécies Reativas de Oxigênio , Superóxidos/metabolismoRESUMO
Analysis of the functional activity of phagocytes is of great importance in the differential diagnosis of patients with recurrent bacterial infections. Here we describe a method to determine the production of reactive oxygen intermediates (ROI) by microcytofluorometry using dihydrorhodamine 123, a derivative of rhodamine 123. Using this method the ROI production of erythrocyte-depleted whole blood samples can be measured without further time-consuming purification steps. Possible harmful manipulation of the isolated cells can also be avoided and highly reproducible and significant results are obtained in the minimum of time. This assay provides a very sensitive alternative to the clinically used NBT test in the diagnosis of patients with chronic granulomatous disease (CGD). Moreover, the analysis of oxygen-dependent effector functions of murine effector cells and cell lines may be important in investigating resistance to certain microbes (e.g., Candida albicans, Staphylococcus aureus or different protozoa such as Toxoplasma gondii or Leishmania species).
Assuntos
Oxigênio/metabolismo , Fagócitos/metabolismo , Rodaminas/farmacologia , Xantenos/farmacologia , Animais , Doença Granulomatosa Crônica/metabolismo , Humanos , Camundongos , Acetato de Tetradecanoilforbol/farmacologia , Zimosan/farmacologiaRESUMO
Deactivation (tachyphylaxis) of the guinea-pig isolated ileum to the spasmogenic action of the complement peptide C5adesArg was analysed. It appeared to consist of 2 components: a fast one, characterized by rapid onset of deactivation and by recovery within 2-3 min (see Damerau et al., 1985b), and a slow component, characterized by progressively increasing loss of sensitivity (until complete deactivation after several minutes) and by recovery within about 80 min. Slow deactivation shows an exponential time course; it is dependent on concentration as well as contact time with C5adesArg and occurs under conditions (incubation in Ca2+-free medium or at 16 degrees C) in which the peptide has no spasmogenic effect. Recovery from slow deactivation follows an exponential time course at 34 degrees C but is blocked at 16 degrees C; on average it reaches about half of the initial sensitivity. The results indicate that the slow deactivation is mainly due to blockade of C5a receptors by the ligand and is independent of the spasmogenic effect of C5adesArg.
Assuntos
Complemento C5/análogos & derivados , Íleo/fisiologia , Músculo Liso/fisiologia , Animais , Complemento C5/fisiologia , Complemento C5a des-Arginina , Cobaias , Técnicas In Vitro , Cinética , Contração MuscularRESUMO
The slow component of deactivation of guinea-pig isolated ileum to C5adesArg was studied to analyse the mechanism of loss and subsequent recovery of sensitivity. Neither cycloheximide (10(-3) M) nor colchicine (5 X 10(-5) M), vinblastine, lumicolchicine, or cytochalasin B (each 2 X 10(-5) M) affected significantly the spasmogenic effect of C5adesArg or the course of deactivation produced by repeated applications; chloroquine (2 X 10(-4) M) inhibited the spasmogenic effect unspecifically without interfering with deactivation. Recovery from slow deactivation was totally blocked by chloroquine and considerably diminished by colchicine and vinblastine, but was not affected by the other agents. It is proposed that recovery involves lysosomal processing of C5a receptors (occupied by the peptide) but does not require biosynthesis of new receptors.