Orientadores: Carmen Silvia Passos Lima, Carmino Antonio de Souza / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-03T15:47:39Z (GMT). No. of bitstreams: 1 Carvalho_PauloVillasBoasde_M.pdf: 1995485 bytes, checksum: 83e53177c0925629f1086f952a6fc5a2 (MD5) Previous issue date: 2003 / Resumo: INTRODUÇÃO: O papel do transplante autólogo de medula óssea (TMO-auto) para o tratamento da leucemia mielóide crônica (LMC) permanece incerto. PACIENTES E MÉTODOS: Onze pacientes com LMC foram indicados para receber o TMO-auto com célula precursora periférica (CPP) mobilizada até seis meses após o diagnóstico, com o mini-ICE (três pacientes) ou a hidroxiuréia (oito pacientes), e obtida por leucaférese. Os pacientes receberam o interferon alfa (IFN) após o TMO-auto. Amostras de medula óssea (MO) obtidas ao diagnóstico e durante o seguimento foram avaliadas por meio da análise citogenética convencional, do método de hibridização ¿in situ¿ com fluorescência (FISH) e por transcrição reversa e reação em cadeia da polimerase (RT-PCR), para a identificação e a quantificação do cromossomo Philadephia (Ph) e do gene BCR-ABL. RESULTADOS: A mediana de seguimento dos pacientes após o TMO-auto foi de 22,7 meses (variação, 0,7-49,1). Um paciente evoluiu para o óbito durante o período de aplasia da MO após a mobilização da CPP. Dez pacientes foram transplantados com CPP com o Ph+ (mediana: 100,0%; variação: 25,0-100,0) e com o gene BCR-ABL (mediana: 68,2%; variação: 27,3-83,5). Um paciente evoluiu para óbito durante a aplasia da MO determinada pelo condicionamento para o TMO-auto. Oito pacientes (88,9%) obtiveram resposta hematológica, sete (77,8%) resposta citogenética, todos (100,0%) obtiveram resposta citogenética molecular por FISH e um paciente (10,0%) obteve resposta molecular por RT-PCR. A mediana das porcentagens de cromossomo Ph, em amostras de MO obtidas seis meses após o TMO-auto (78,0%), foi menor do que a observada ao diagnóstico (100,0%; P=0,035). Foram também menores as medianas das porcentagens de núcleos interfásicos com o gene BCR-ABL em amostras de MO obtidas três, seis e nove meses após o TMO-auto (4,0%, 7,3% e 15,7%, respectivamente), em comparação a observada ao diagnóstico (82,5%; P<0,050). Ao final do estudo, nove pacientes estavam vivos, em fase crônica da doença, sendo que quatro deles apresentavem respostas hematológica, citogenética e citogenética molecular. CONCLUSÃO: O TMO-auto em associação com o IFN possibilita a obtenção de respostas hematológica, citogenética, citogenética molecular e molecular para pacientes com fases iniciais da LMC / Abstract: INTRODUCTION: The role of the autologous stem cell transplantation (ASCT) as a treatment procedure for chronic myeloid leukaemia (CML) patients remains uncertain. PATIENTS AND METHODS: Eleven CML patients were indicated to receive ASCT with peripheral blood progenitor cells (PBPCs) mobilised within six months from diagnosis with mini-ICE (three patients) or hydroxyurea (eight patients) and obtained by leukapheresis. The interferon alpha (IFN) was administered to patients after ASCT. Bone marrow (BM) samples obtained at diagnosis and during evaluations after ASCT were analysed by cytogenetics, fluorescence ¿in situ¿ hybridisation (FISH) and reverse transcription-polimerase chain reaction (RT-PCR), with the purpose of identifying and quantifying the Philadelphia chromosome (Ph+) and the BCR-ABL gene. RESULTS: The median follow-up of patients after ASCT was 22.7 months (range: 0.7-49.1). One patient died during the aplastic period determined by the mobilisation regimen of PBPCs. Ten patients received ASCT with PBPCs with Ph+ (median: 100.0%; range: 25.0-100.0) and FISH+ cells (median: 68.2%; range: 27.3-83.5). One patient died during the aplastic phase due to BM conditioning regimen. Eight patients (88,9%) achieved haematological response, seven (77.8%), all of them (100.0%) molecular cytogenetics response by FISH, and one unique patient (10,0%) achieved molecular response by RT-PCR. The median percentage of Ph metaphases in BM samples obtained after three months of ASCT (78.0%) was lower than the percentage obtained at diagnosis (100,0%; P=0.035). The median percentages of FISH+ interphase nuclei obtained after three, six, and nine months after ASCT (4.0%, 7.3% and 15.7%, respectively) were also lower than that obtained at diagnosis (82,5%; P<0.050). At the end of the study, nine patients were alive, in chronic phase of CML. Four of them presented haematological, cytogenetics, and cytogenetics responses. CONCLUSION: The ASCT associated with IFN therapy results in haematological, cytogenetics, molecular cytogenetics and molecular responses in early chronic phase of CML¿s patients / Mestrado / Mestre em Clinica Medica

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The interferon (IFN) system is a powerful antiviral defense system. Host cell pattern recognition receptors (PRRs) recognise pathogen-associated molecule patterns (PAMPs) which when activated, lead to the transcription of the IFN-β gene. As a consequence IFN is secreted from the cell and activates the JAK-STAT pathway to up-regulate the transcription of IFN-stimulated genes (ISGs). The products of many ISGs inhibit viral replication and cell proliferation. Viruses encode IFN antagonists that dampen down the IFN response, making it less effective. However, within a virus population, there are always likely to be naturally occurring mutant viruses that have lost the ability to circumvent the host IFN response, and if isolated, these viruses would be unlikely to cause severe disease in the host and may therefore be developed as live attenuated virus vaccine candidates. To develop a methodology to rapidly isolate IFN-inducing mutant viruses, we generated an A549 reporter cell-line in which expression of GFP was driven by the IFN-β promoter. Using this cell-line, we show that the number of cells that became positive for GFP correlated with the amount of IFN secreted by the infected cells and the number of defective interfering (DI) particles within the virus preparations. However, we were unable to isolate IFN-inducing mutant viruses using the A549/pr(IFN-β).GFP cell-line(s). Possible reasons for this may be either that, in cells infected by IFN-inducing mutant viruses, an antiviral state was established independent of IFN that prevented virus replication in the reporter cells in which the IFN-β promoter was activated; or the viruses that activated the IFN-β promoter were DIs only which were not be able to replicate without non-defective helper viruses. A549/pr(IFN-β).GFP cells are also being used for high throughput assays to screen chemical libraries for compounds that block IFN induction. Such compounds may be potential candidates for anti-inflammatory drugs.

Polymorphisms in the interferon regulatory factor 5 (IRF5) gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE). IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s), it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1) SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2) an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3) an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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