published_or_final_version / Paediatrics / Master / Master of Philosophy

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [58]-87).

The growth of B-cell lymphoma, LSCC-RP9, in culture was inhibited by spleen cells from bursa-immunized agammaglobulinemic (A-gamma) chickens. This inhibition was mediated by suppressor T-cells. The growth of transplantable LSCT-RP6 B-cell lymphoma was suppressed in A-gamma chickens, while that of the control SPCT-RP11 T-cell tumor was not affected. Furthermore, the incidence and growth of the LSCT-RP6 tumor in normal recipients were decreased when it was co-transplanted with spleen cells from bursa immunized A-gamma chickens. The results suggest that suppressor T-cells inhibit the growth of B-cell lymphoma.

Orientador: Maria Marluce dos Santos Vilela / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T09:35:25Z (GMT). No. of bitstreams: 1 Ramalho_VanessaDomingues_M.pdf: 1157730 bytes, checksum: 0cd719050713e1b2340be47d4d5f5b38 (MD5) Previous issue date: 2010 / Resumo: A agamaglobulinemia ligada ao X (XLA; OMIM#300755) é uma imunodeficiência primária humoral caracterizada por um bloqueio na diferenciação dos linfócitos B na medula óssea, levando à profunda hipogamaglobulinemia e reduzido número ou ausência de células B periféricas. Os pacientes com XLA são susceptíveis a infecções recorrentes por bactérias encapsuladas e enterovírus devido à deficiência de anticorpos. Mutações no gene codificante da tirosina quinase de Bruton (Btk) são responsáveis pela doença. Btk é uma tirosina quinase citoplasmática da família Tec importante no desenvolvimento, na diferenciação e na sinalização dos linfócitos B. A detecção de mutações no gene btk possibilita o diagnóstico definitivo de XLA. O objetivo deste estudo foi identificar e caracterizar mutações em btk. Foram incluídos 6 pacientes conforme os critérios do PAGID e ESID: indivíduos do sexo masculino com menos de 2% de linfócitos B periféricos, hipogamaglobulinemia e história de infecções bacterianas de repetição. A triagem de mutações foi realizada com a técnica de SSCP e possíveis mutações foram confirmadas por seqüenciamento. A expressão de Btk nos pacientes e mães foi avaliada em monócitos por citometria de fluxo. Dentre os pacientes analisados as principais manifestações clínicas foram as infecções do trato respiratório. Todos tiveram início dos sintomas durante o primeiro ano de vida, linfócitos B periféricos abaixo de 2% e hipogamaglobulinemia anterior ao início da terapia de reposição de imunoglobulinas. Foram identificadas cinco mutações em btk, três novas (p.Ala347fsX55, p.I355T e p.Thr324fsX24) e duas já descritas na literatura (p.Q196X e p.E441X). A detecção das mutações nos pacientes permitiu a análise mutacional de mães, avós e tias maternas. Três mães e uma avó foram confirmadas portadoras de XLA. Em adição, os valores de expressão de Btk obtidos mostraram deficiência da proteína (4,5% a 65,2%) nos pacientes e um padrão bimodal de expressão de Btk foi observado nas mães, indicando o estado de portadora de XLA. Em um dos pacientes não foi identificada mutação, entretanto a expressão de Btk mostrou-se reduzida. O uso combinado da análise genética e da avaliação da expressão de Btk por citometria de fluxo possibilitou o diagnóstico definitivo de XLA e a identificação de portadoras da doença. / Abstract: X-linked agammaglobulinemia (XLA; OMIM# 300755) is a primary humoral immunodeficiency characterized by a block in early B cell differentiation, leading to profound hypogammaglobulinemia and few or no circulating B cells. Patients with XLA are susceptible to recurrent infections by encapsulated bacteria and enteroviruses due to antibody deficiency. Mutations in the Bruton tyrosine kinase (Btk) gene have been identified as responsible for XLA. Btk is a cytoplasmic tyrosine kinase of the Tec family important in B-lymphocyte development, differentiation, and signaling. Detection of a btk mutation allows definitive diagnosis of XLA. The aim of this study was to identify and characterize mutations in btk. Six patients were included according to the criteria of PAGID and ESID: males with less than 2% of circulating B cells, hypogammaglobulinemia and a history of recurrent bacterial infections. Mutation screening was performed with SSCP technique and possible mutations were confirmed by sequencing. Expression of Btk protein in patients and mothers was assessed in monocytes by flow cytometry. The major clinical manifestations among patients were respiratory tract infections. All had onset of symptoms during the first year of life, circulating B cells below 2% and hypogammaglobulinemia before the start of immunoglobulin replacement therapy. We identified five mutations in btk, three novel (p.Ala347fsX55, p.I355T and p.Thr324fsX24) and two recurrent mutations (p.Q196X and p.E441X). The btk mutations detection in patients enabled the screening of mothers, grandmothers and maternal aunts. Three mothers and one grandmother were confirmed XLA carriers. In addition, flow cytometric evaluation of Btk expression in monocytes revealed that Btk deficiency (4,5% a 65,2%) was present in patients and a bimodal pattern of Btk expression was observed in mothers, indicating that they were XLA carriers. In one patient no mutation was identified, but his Btk expression was reduced. The combined use of genetic analysis and flow cytometric assay of Btk protein expression allowed the definitive diagnosis of XLA and its carriers detection. / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente

Orientador: Maria Marluce dos Santos Vilela / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T17:02:47Z (GMT). No. of bitstreams: 1 Ramalho_VanessaDomingues_D.pdf: 1441004 bytes, checksum: a45d3f4f8825288e2959115ef25fc13a (MD5) Previous issue date: 2014 / Resumo: A agamaglobulinemia ligada ao X (XLA; OMIM#300755) é caracterizada por um bloqueio na diferenciação dos linfócitos B na medula óssea, levando à profunda hipogamaglobulinemia e reduzido número ou ausência de linfócitos B periféricos. Os pacientes são susceptíveis a infecções recorrentes por bactérias encapsuladas e enterovírus. XLA é causada por mutações no gene da tirosina quinase de Bruton (BTK). Contudo, não há estudos de relação entre expressão protéica e o tipo de mutação, nem sobre as conseqüências da retenção intracelular do excesso de proteínas mal formadas. Os objetivos deste trabalho foram avaliar a expressão de BTK e sua relação com o tipo de mutação em pacientes com XLA, assim como verificar suas conseqüências nos sensores de estresse do retículo endoplasmático. O diagnóstico de XLA foi baseado em infecções recorrentes, níveis significativamente reduzidos de IgM, IgG e IgA, linfócitos B circulantes <2% e mutação identificada no gene BTK. A expressão dos transcritos de BTK foi avaliada por PCR quantitativo em tempo real em oito pacientes XLA e oito controles. Pela mesma técnica, foi avaliada a expressão de 10 genes do estresse do retículo endoplasmático em seis pacientes e seis controles. Foram caracterizadas quatro mutações missense, uma mutação nonsense, dois frameshifts e um defeito em sítio de splicing. As mutações do tipo nonsense, frameshift e defeito em sítio de splicing levaram à formação de stop codon prematuro. Foi detectado um perfil de expressão de BTK diferenciado nos pacientes com mutações com stop codon prematuro em comparação aos pacientes com mutações missense e controles saudáveis. Especificamente, os pacientes com mutações com stop codon prematuro apresentaram redução da expressão de BTK (P = 0,004). No entanto, verificamos que as mutações missense não afetaram a expressão de BTK. Por meio de imunocitoquímica, encontramos que as mutações com stop codon prematuro levaram à deficiência da expressão da proteína BTK e as do tipo missense resultaram na localização anormal da proteína no citoplasma celular, o que evidencia a síntese de proteína não funcional. Os pacientes com XLA apresentaram expressão aumentada do marcador de estresse do retículo endoplasmático XBP1 (P = 0,002). Em conclusão, a quantificação da expressão de mRNA para BTK é uma ferramenta para diferenciar as conseqüências mutacionais em pacientes com XLA. Ela também pode contribuir para o estudo de transcritos em outras doenças genéticas com diferentes tipos de mutação. Este é o primeiro relato de estresse do retículo endoplasmático na agamaglobulinemia ligada ao X / Abstract: X-linked agammaglobulinemia (XLA, OMIM # 300755) is characterized by a block in differentiation of B lymphocytes in the bone marrow, leading to profound hypogammaglobulinemia and few or no peripheral B lymphocytes. Patients are susceptible to recurrent infections by encapsulated bacteria and enteroviruses. XLA is caused by mutations in the Bruton tyrosine kinase gene (BTK). However, there have been no studies on the relationship between protein expression and the type of mutation, nor on the consequences of the disruption of protein folding that results in intracellular retention. The objectives of this study were to evaluate BTK expression and its mutation type in patients with XLA, as well as to verify their consequences on the endoplasmic reticulum stress sensors. The XLA diagnosis was based on recurrent infections, significantly reduced levels of IgM, IgG and IgA, circulating B lymphocytes <2% and BTK gene mutation identified. The expression of BTK transcripts was assessed by quantitative real-time PCR in eight XLA patients and eight control subjects. By the same technique, the expression of 10 endoplasmic reticulum stress genes was measured in six patients and six controls. Four missense mutations, one nonsense mutation, two frameshifts and a splice site defect were characterized. Mutations of the nonsense type, frameshift and splice site defect led to a premature stop codon formation. A differential profile of expression of BTK was detected in patients with mutations that led to a premature stop codon compared to patients with missense mutations and healthy controls. Specifically, patients with mutations resulting in a premature stop codon exhibited reduced expression of BTK gene (P = 0.004). However, it was found that missense mutations did not affect BTK expression. By immunocytochemistry, we found that mutations with a premature stop codon impaired expression of BTK protein and that missense mutations led to an abnormal localization of the protein in the cell cytoplasm, showing the synthesis of a non-functional protein. Patients with XLA showed increased expression of the endoplasmic reticulum stress marker XBP1 (P = 0.002). In conclusion, the quantification of mRNA expression for BTK is a tool to differentiate mutational consequences in patients with XLA. It can also contribute to the study of transcripts in other genetic diseases with different types of mutation. This is the first report on endoplasmic reticulum stress in X-linked agammaglobulinemia / Doutorado / Saude da Criança e do Adolescente / Doutora em Ciências

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Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH SUMMARY: Background: Primary immunodeficiency disorders (PID) disrupt the proper functioning of the immune system. The prototypic PID is X-linked Agammaglobulinemia (XLA). This disorder is caused by mutations in the Bruton tyrosine kinase (Btk) gene and results in an arrest in B cell development which leads to a profound reduction of all classes of serum immunoglobulins (i.e antibodies). Patients with a lack of antibodies experience recurring bacterial infections during early childhood that can be fatal if not treated. Intravenous gammaglobulin replacement therapy (IVIg) is the standard treatment for XLA. It provides passive immunity thereby reducing the number and severity of infections as well as limiting many of the infectious complications. Early detection and treatment of XLA allows affected individuals to live a relatively normal life. Objective: The purpose of this study was to determine the molecular basis of XLA in South Africa using a direct sequence-based method to detect abnormalities in the Btk gene to aid clinical diagnosis of the disease. Methods : Male patients with a clinical diagnosis of XLA were included in this study. Genetic analysis was used to explore the exonic region of the Btk gene of 5 unrelated male patients and compared to 10 healthy controls. Family members were followed up to determine carrier status, where possible. Results: Mutational analysis revealed Btk abnormalities in 4 of the 5 patients leading to a definitive diagnosis of XLA. Two of the three mutations found in this study have been previously described while one mutation appears to be novel. The novel mutation is a one base pair deletion in exon 16 which leads to the truncation of the Btk protein. Despite the clinical findings suggestive of XLA, no mutation was identified in the exonic region of the Btk gene of the remaining patient, indicating that this patient might have a different form of PID. Maternal follow-up confirmed the maternal inheritance pattern as all mothers screened were carriers of the Btk mutation present in the affected individual. Discussion : Using a direct sequence-based method abnormalities were identified in the Btk gene of three patients. Molecular diagnosis coupled to clinical history of the patient provides a definitive XLA diagnosis. This study supports the use of molecular techniques in the diagnosis of PID and underlines the synergy that could be possible in a clinical setting. / AFRIKAANSE OPSOMMING: Agtergrond: Primêre immuungebrek siektes (PIGS) word gekenmerk aan ‘n gebrek aan teenliggame in die immuunsisteem wat lei tot herhaalde infeksies in jong kinders wat fataal kan wees indien dit nie vroegtydig behandel word nie. Die prototype van die bekende PIGS is X-gekoppelde Agammaglobulinemia (XGA). Die siekte word veroorsaak deur mutasies in die Bruton Tirosien kinase (Btk) geen en lei tot ʼn stilstand in B sel ontwikkeling en gevolglik ʼn vermindering van alle klasse van serum immuunoglobulins (teenliggaam). Intraveneuse gammaglobulien vervangingsterapie(IVIg) is die standaard behandeling vir XGA. Dit voorsien passiewe immunitiet en gevolglik verminder dit die getal en erns van infeksies en beperk baie van die aansteeklike komplikasies. Vroeë diagnose en behandeling van XGA laat toe dat geaffekteerde individue ʼn relatiewe normale lewe ly. Doel: Die doel van hierdie studie is om die molekulêre basis van XGA in Suid Afrika te ondersoek, deur gebruik te maak van direkte volgorde bepaling van die Btk geen in die hoop om die kliniese diagnose van die siekte aan te help. Metode : Manlike pasiente met ‘n kliniese diagnose wan XGA was by die studie ingesluit. Genetiese analise was gebruik om die “exonic” omgewing van die Btk geen te ondersoek van 5 onverwante manlike pasiente en vergelyk teenoor 10 gesonde kontrole. Waar moontlik was familie lede ogevolg om draers te bepaal. Resultaat: Mutasies in die Btk geen is geidentifiseer in 3 van die 4 pasiente, klinies gediagnoseer meet XGA. Die mutasies sluit 2 reeds beskryfde variante in en een nuwe mutasie, ‘n een basis paar delesie in ekson 16 van die Btk geen, Ten spyte van die kliniese profiel suggestief van XGA in die 5de pasient, was geen mutasies geidentifiseer in die “exconic” omgewing van die Btk geen nie, dit kan moontlik toegeskryf word aan die teenwoordigheid van ‘n ander vorm van PIGS in hierdie pasient. Opvolg analise op die DNA van die moeders van die pasiente het die moederlike oorerwings patroon van die siekte bevestig aangesien al die moeders draers van die geidentifiseerde mutasie in die Btk geen van die gaffekteerde individu was. Gevolgtrekking: Genetiese analise van die Btk geen blyk ʼn sensitiewe en spesefieke metode te wees om individue met XGA te diagnoseer. Hierdie studie ondersteun die gebruik van molekulêre metodes in die diagnose van PIGS en beklemtoon die moontlike sinergie wat kan bestaan tussen hierdie tipe benadering in die kliniese omgewing. / National Research Foundation / National Health Laboratory Services : Pathology Research Development Grant of NHLS Research Trust Grants

In comparison to men, the number of X-linked genes is doubled in women as they have two chromosomes X while men are hemizygotes for X-linked genes. This imbalance is compensated by X inactivation (XCI) process, also known as primary X-inactivation, occurring in the early stage of embryogenesis. X inactivation is a random process and females are mosaics of two cell populations. The ratio of expressed alleles in women can be random (50:50) or skewed (≥80:20). The skewed X inactivation may occur due to selection when one of the alleles is preferentially inactivated (secondary X inactivation). In this study XCI status in heterozygous females with various severity of phenotypic symptoms and traits in selected X linked inherited metabolic diseases is analysed, with the focus being Fabry disease - the deficiency of the enzyme alpha-galactosidase A encoded by GLA gene. Moreover, XCI in one family with X linked agammaglobulinemia is examined. Mutant alleles and XCI status based on various loci, different methodical approaches and different tissues is subjected to examination. For the first time, the direct analysis of GLA gene transcript to detect the allele ratio was used alongside with the single-nucleotide polymorphisms in the IDS and LAMP genes for allele-specific expression (ASE) and the AR, RP2 and...