No description available.

Thesis (M. Phil.)--University of Hong Kong, 1997. / 3 articles written by the author in pocket. Includes bibliographical references (leaf 154-186).

published_or_final_version / Paediatrics / Master / Master of Philosophy

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met een samenvatting in het Nederlands.

No description available.

Abstract available in the Journal of Investigative Medicine.

Neutrophils playa key role in the systemic inflammatory response which may lead to serious tissue injury and multiple organ dysfunction. In this setting, activated neutrophils, largely in response to tumour necrosis factor-alpha (TNF-α), secrete reactive oxidants, granule proteases and bioactive lipids, as well as pro-inflammatory cytokines, emphasising the importance of these cells as targets for anti-inflammatory therapies. There are, however, only a few currently available agents that directly modulate neutrophil pro-inflammatory responses in clinical practice, with corticosteroids being relatively ineffective against these cells. Although, the anti-inflammatory potential of cAMP-elevating agents has been recognised, the exact molecular/biochemical mechanisms which underlie the anti-inflammatory actions of epinephrine and related β-agonists with neutrophils, have not been established. Epinephrine treatment of neutrophils resulted in increased intracellular cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, further supporting a cAMP-mediated effect. Although epinephrine did not affect the release of Ca2+ from neutrophil intracellular stores, the rate of clearance of cytosolic Ca2+ was accelerated by this agent. In the setting of decreased efflux and a reduction in store-operated influx of Ca2+, these effects of epinephrine are compatible with enhancement of the cAMP-dependent Ca2+ sequestering/resequestering endo-membrane Ca2+-ATPase. Epinephrine therefore down-regulates the pro-inflammatory activation of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca2+. Comparison of the effects of 4 selective (fenoterol, formoterol, salbutamol and salmeterol) and 3 non-selective (epinephrine, norepinephrine and isoproterenol) β-adrenoreceptor agonists, on the pro-inflammatory activities of human neutrophils, demonstrated that the agents tested clearly differ with respect to anti-inflammatory potential. Epinephrine, isoproterenol, fenoterol and formoterol significantly increased intracellular concentrations of cAMP in neutrophils, an activity which was paralleled by inhibition of the production of reactive oxidants and release of elastase from FMLP-activated cells. Salbutamol and salmeterol on the other hand, did not cause significant suppression of the pro-inflammatory activities of these cells. The effect of norepinephrine was intermediate between these two groups. The inhibitory effects of βagonists are mediated via β2-adrenergic receptors on the neutrophil membrane. The relationship between activation of NAOPH oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from 4 subjects with chronic granulomatous disease (CGO). Activation of CGO neutrophils was accompanied by a prolonged increase in cytosolic Ca2+, occurring in the setting of trivial membrane depolarisation and accelerated influx of Ca2+. This was associated with hyperactivity of the cells with excessive elastase release, which was attenuated by the type 4 phosphodiesterase inhibitor, rolipram. These findings support the involvement of NAOPH oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses, and granuloma formation, which are characteristic of CGO. Store-operated influx of Ca2+ into activated neutrophils is stringently regulated, presumably to prevent hyperactivation of the cells. The major contributors to this physiologic, anti-inflammatory process are NAOPH oxidase which, by its membrane depolarising actions excludes extracellular Ca2+, and the plasma membrane and endomembrane Ca2+-ATPases, which mediate clearance of store-derived cation. Subsequent influx of the cation, through store-operated Ca2+ channels is controlled by the relatively slow, restraining, membrane repolarising action of the Na+/Ca2+ exchanger, enabling efficient diversion of incoming cation into stores. / Thesis (DPhil (Immunology))--University of Pretoria, 2005. / Immunology / unrestricted

Orientador: Antonio Condino Neto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T01:07:19Z (GMT). No. of bitstreams: 1 AgudeloFlorez_PiedadMatilde_D.pdf: 7673448 bytes, checksum: 14c8d7fc8437933b652076b21b9f6ff6 (MD5) Previous issue date: 2004 / Resumo: Doença Granulomatosa. Crônica (DOC) é uma imunodeficiência caracterizada por infecções recorrentes graves. Os defeitos moleculares que lavam a DGC são geralmente devidos a mau funcionamento, ausência o baixa expressão de um dos componentes do sistema NADPH oxidase. Este trabalho analisou o uso de RT-PCR para a triagem de defeitos moleculares responsáveis por DGC ligada ao X em 8 pacientes. RNA total foi preparado de linfócitos B transformados com vírus de Epstein-Barr e transcrição reversa com hexâmeros randomicos. O cDNA resultante foi amplificado por PCR com oligonucleotídeos específicos para 3 regiões exônicas abrangendo toda a extensão do gene. Com esta estratégia foi possível a detecção da expressão defeituosa de gp91-phox em 7 pacientes. Concluímos que a análise por meio de RT-PCR, um método alternativo menos complexo, rápido e econômico foi apropriado para detecção inicial de defeitos moleculares em 7 de 8 pacientes com DOC ligada ao X. Posteriormente investigamos em detalhe os defeitos genetico-moleculares de 7 crianças não relacionadas com DGC ligada ao X. Todos os pacientes foram procedentes do Chile e Brasil. Encontramos uma inserção c.1267_1268insA no paciente JY no exon 10 levando a uma mutação tipo "&ameshill:". Esta mutação é um novo registro na literatura. Detectamos duas substituições "nonsense", uma no paciente PT, c.95 G>A no exon 2 que leva a um códon de parada W28X e outra no paciente MF, c.229 C>T no exon 3 que leva a um códon de parada R73X. Em 4 casos, nos pacientes IC, Vin, RS, GO, diferentes erros de "splicing" foram encontrados. Dois pacientes apresentaram uma subtitução c.264 G> A ao final do exon 3. Os dois restantes apresentaram uma subtitução c.1326 + 1 G>A no intron 10 e outra subtitução c.1164 - 2 A>O no intron 9. Esta última mutação também é um novo registro na lit_ratura. As mutações identificadas na proteína gp91-phox confirmam um alto grau de heterogeneidade molecular como é relatado em outros grupos étnicos e a importância de investigar os defeitos moleculares em diferentes populações. Contrastando com esta heterogeneidade, a DGC associada com defeitos na proteína p47-phox, apresentam pouca variabilidade. Neste estudo, os pacientes analisados, dois irmãos, mostram uma deleção homozigota OT (L}.GT) no começo do exon 2. L Também é analisado o caso de um paciente com infecções recorrentes que inicaJmente recebeu o diagnóstico de deficiência de G6PD. Estudos moleculares mostraram que a deficiência de G6PD foi devida a uma mutação 202 G_A, variante Afi:icana. . O paciente também mostrou uma reduzida atividade da explosão respiratória como observado em DGC ligada ao X. A análise do gDNA mostrou uma subtitução 264 G_A na região do splicing do exon 3 da proteína gp91-phox. A seqüência do cDNA detectou uma deleção do exon 3, levando a uma mutante inestável ou não funcional da proteina gp91-phox e resultando no fenótipo de DGC ligada ao X. Propomos que ftente a um paciente com deficiência de G6PD com episódios de infecções graves considerem a possibilidade de um defeito na atividade fagocítica e uma eventual associação com DGC / Abstract: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency characterized by ear1y onset recurrent severe infections. The molecular defects causing CGD are generally due to the absence, low expression or malfunctioning of one of the NADPH oxidase components. This work analyzed the potential use of reverse transcription (RT)PCR for screening molecular defects responsible for X-linked CGD in 8 Brazilian patients. Total RNA was prepared from EBV-transformed B-lymphocytes and reverse transcribed using random hexamers. The resultant cDNA was PCR-amplified by specific and overlapping pairs of primers regarding 3 exonic regions of gp91-phox gene. This strategy made possible the detection of defective gp91-phox expression in 7 patients. We conclude that RT -PCR analysis, a less complex, more economic and faster alternative method, was appropriate for screening molecular defects in 7 out 8 X-linked CGD patients. We further investigated the molecular genetic defects in 7 unrelated patients with X-linked CGD, from Chile and Brazil. We found an insertion c.1267_1268insA in exon 10 leading to a frameshift mutation. This mutation is a novel reporto we detected two single base-pair substitutions that lead to nonsense mutations. The first was a c.95 G>A substitution in the exon 2 which predicts a stop codon W28X and the second was a c.229 C> T substitution in the exon 3 which predicts a stop codon R73X. We also identified different splice site mutations in 4 cases. Two patients presented a c.264 G> A substitution at the end of exon 3. The remaining two patients presented either a c.1326 + 1 G>A substitution in intron 10 or a c.1164 - 2 A>G substitution in intron 9. This Iast mutation is also novel. The gp91-phox mutations identified in these patients show a high degree of molecular heterogeneity as reported in other ethnic groups and the importance to investigate molecular genetic defects in diferent populations. Contrasting with the heterogeneity of mutations observed in X-linked CGD, the disease associated with defect in p47-phox shows less variability. In this report, the patients with CGD, two sib1ings, show a homozygotous dinucleotide GT deletion (.6.GT) at the beginning of exon 2. We also reported a child with recurrent infections who initially received the diagnosis of G6PD deficiency. Molecular studies showed that the G6PD deficiency was due a 202 G-+A mutation, the A- variant common in African ethnic groups. The proband also exln'bited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G-+A substitution at the 3' splice junction of gp91-phox exon 3. The cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable or nonfunctional mutant gp91-phox and to the phenotype of Xlinked CGD. We propose that clinicians in face of a patient with G6PD deficiency under a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes microbicidal activity, and the eventual association of G6PD deficiencyand chronic granulomatous disease / Doutorado / Saude da Criança e do Adolescente / Doutor em Saude da Criança e do Adolescente

Rationale: CGD is characterized by recurrent life-threatening infections with bacterial and/or fungal pathogens and granuloma formation. This is caused by defects in the phagocyte NADPH oxidase systems. Deficiency of MBL caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. Combined deficiencies of MBL and NADPH oxidase have not been described commonly, and we report one such case. Methods: The patient records were reviewed, and laboratory data collected. Genetic mutation analysis for MBL2 gene was done at IBT laboratory while CGD mutation analysis is pending. Results: A 2 y/o Caucasian male presents with an intermittent fever for several weeks with an enlarging blister in the right anterolateral thigh. The patient had a history of recurrent pneumonias. Chest CT demonstrated prominent interstitial markings with pulmonary nodules. Lung Biopsy revealed multifocal nodular necrotizing granulomas. Neutrophil Oxidative Burst Test was consistent with X-linked CGD (Patient 14.3/Control 1375.5). The serum level of MBL was 25ng/mL (N=>500). MBL genetic analysis showed LXPA/LYPB (the latter being the defective haplotype). Elevated CRP and polyclonal hyperglobulinemia were consistent with previous report in CGD. The patient was treated with itraconazole, bactrim and interferon gamma with consistent improvement. Conclusions: The role of MBL deficiency in this patient with severe necrotizing granulomatous disease of the lung is unclear. However it seems likely that MBL defects will contribute to worse infections in patients with phagocyte malfunction. The true prevalence of MBL defect in CGD needs to be studied.

No description available.