Dissertations / Theses on the topic 'Interferony (IFNy)'

Dendritic cells (DC) and macrophages are mononuclear phagocytes present in the tissues of the anogenital tract where HIV-1 transmission occurs in almost all cases. These cells are both target cells for HIV-1 and act as the primary opportunity for the virus to inhibit the innate recognition. Our lab has previously shown that both cell types fail to produce type I interferons (IFN) in response to HIV-1. However, it stimulates them to produce specific subsets of interferonstimulated genes (ISGs) some of which have direct antiviral activity. Our lab defined the precise stage in the IFN inducing signalling pathway that HIV-1 targets; blocking the phosphorylation but not K63 polyubiquitination of TANK-binding kinase 1 (TBK1). Finally, we showed that this is mediated by two HIV-1 accessory proteins, Vpr and Vif. In the first results chapter (3) of this thesis, we show that ISG expression is governed by two phases: an initial induction through the detection of the incoming HIV-1 inoculum at 1-6 hpi and then a second phase predominantly by newly transcribed HIV-1 as observed after 72 hpi. We also show that knockdown of either Tat, IRF1 or IRF7 ablated the second phase of ISG subset and that Tat/IRF7 induction of the second phase ISGs is down stream of IRF1. In the second results chapter (4), we demonstrate that Vpr (but not Vif, Nef or Vpu) is the only accessory protein observed to co-immunoprecipitate with TBK1 in the absence of other viral proteins. This was shown using two different Co-IP assay methods (G-Sepharose beads and the Trap system). Therefore, this supports our hypothesis that HIV-1 inhibits IFN signalling in mononuclear phagocytes by physical interactor of its protein with TBK1, thereby blocking its functional role in IFN signalling. Defining the minimal binding regions TBK1 and Vpr interaction could allow the development of small molecule inhibitors of the interaction that would rescue IFNβ production in these cells and reduce HIV replication and spread.

Orientadores: Leonilda Maria Barbosa Santos, Ilma Aparecida Paschoal
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Mestrado
Imunologia
Mestre em Ciências Biológicas

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
A hansenÃase, cujo agente etiolÃgico à o Mycobacterium leprae, à doenÃa de amplo espectro clÃnico e imunopatolÃgico. Suas apresentaÃÃes clÃnicas estÃo correlacionadas com padrÃes imunolÃgicos distintos, variando de uma vigorosa resposta imune mediada por cÃlulas ao M. leprae, com padrÃo tipo 1 no polo tuberculÃide, a uma ausÃncia de resposta celular especÃfica aos antÃgenos do M. leprae no pÃlo lepromatoso, com predomÃnio de resposta tipo 2 e exacerbaÃÃo da resposta humoral. A capacidade do antÃgeno bruto de M. leprae em estimular cÃlulas mononucleares do sangue perifÃrico (PBMC) na produÃÃo de IFN-y foi avaliada em pacientes com hansenÃase e em seus contactantes, do municÃpio de Sobral-CE. Um total de 30 casos foi estudado, antes de receberem tratamento poliquimiterÃpico. O grupo de casos paucibacilares foi constituÃdo por oito com a forma indeterminada, dez com a forma tuberculÃide, dois com a forma dimorfo tuberculÃide; e o grupo multibacilar foi constituÃdo por dez com a forma virchoviana e dois com a forma dimorfa virchoviana. O grupo de contactantes foi constituÃdo por sessenta indivÃduos, sendo 1 consangÃÃneo e 1 nÃo consangÃÃneo para cada caso Ãndice. O antÃgeno bruto de M. leprae estimulou a produÃÃo de IFN-y nas PBMC de sete casos dimorfo tuberculÃide/tuberculÃide (DT/TT), trÃs com a forma indeterminada e dois com a forma dimorfa virchoviana/virchoviana (DV/VV). O grupo DT/TT produziu nÃveis de IFN-y significantemente maiores que o grupo DV/VV (Teste de Fisher, p=0,027). A produÃÃo de IFN-y nos contactantes foi observada em 34 indivÃduos, 21 consangÃÃneos e 13 nÃo consangÃÃneos. NÃo foi observada diferenÃa significativa entre os contactantes do grupo paucibacilar (forma indeterminada, DT/TT) e multibacilares (DV/VV). PorÃm, foi observada diferenÃa significativa na produÃÃo desta citocina entre contactantes e casos DV/VV. O estudo sugere que nÃo hà diferenÃa significativa na produÃÃo de IFN-y entre indivÃduos contactantes consangÃÃneos e nÃo consangÃÃneos, dos casos paucibacilares e multibacilares. AlÃm disso, ao correlacionarmos a produÃÃo desta citocina nos indivÃduos com a presenÃa de cicatriz, tambÃm nÃo observamos diferenÃa significativa
Leprosy, which is caused by Mycobacterium leprae, is an illness of ample clinical and immunopathological spectrum. Its clinical manifestations are correlated with distinct immunologic form, varying from a vigorous immune response mediated by cells to M. leprae, with type 1 standard in the tuberculÃide polar region, to an absence of specific cellular response to antigens of M. leprae in the lepromatous polar region, with predominance of type 2 response and exacerbations of humoral response. The capacity of whole M. leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) in the IFN-y production was measured in leprosy patients and their householdâs contacts, in the city of Sobral, state of CearÃ. A total of 30 leprosy patients were used for the study, before start chemotherapy. The paucibacilary leprosy patient group consisted of eight polar indeterminate, ten polar tuberculÃide, two borderline tuberculÃide, and the multibacilary leprosy patient group consisted of ten lepromatous leprosy and two borderline lepromatous leprosy. The household contacts group consisted of sixty healthy individuals, consanguineous and non consanguineous. The whole M. leprae antigen stimulated IFN-y production in the PBMC of seven borderline tuberculÃide/tuberculÃide (DT/TT), three indeterminate form, two borderline lepromatous/lepromatous (DV/VV the DT/TT group produced IFN-y levels significantly higher than DV/VV group (Fisher Test, p=0,027). The IFN-y production in the household contacts was observed in 34, 21 consanguineous and 13 non consanguineous. It wasnât observed significant difference between paucibacilary householdâs contacts (DT/TT, indeterminate form) and multibacilary householdâs contacts groups in the IFN-y production to whole M. leprae antigen. However, it was observed significant difference in the production of these cytokine between household contacts and DV/VV patients. This study suggests that there wasnât significative difference in the production IFN-y between non consanguineous and consanguineous subjects of the paucibacilary and multibacilary cases. Moreover, at correlacionated the production in these cytokine in the subjects with the presence of scar, we didnât observed significative difference too

Interferon beta-1a (IFNβ-1a) is a recombinant IFNβ with the tradename Rebif involved in several biological activities. Recently, it has been reported that artificial deamidation of IFNb-1a increases its biological response. Given the therapeutical potential, an investigation on the deamidated variant has been carried out via different approaches to discover the mechanism underlying this biological effect. The antiviral and immunomodulatory activity of deamidated cytokine was assessed using two precise and accurate cell-based assays. As expected, deamidated IFNβ-1a showed an increase in the biological response and its canonical pathway and receptor binding affinity were thorough analysed. Deamidated interferon beta increases STAT1 phosphorylation and ISGs expression compared to its native form. A full in-depth analysis in receptor binding highlighted a change in receptor affinity in deamidated IFNβ-1a. In particular, deamidation destabilizes the interaction with IFNAR2 through a change in the H-bonds network, increasing the affinity to IFNAR1 and consequently to the whole receptor complex. The higher receptor binding and the consequent strong signaling and gene expression, explains the greater biological activity of the deamidated IFNβ-1a compared to the native fcorm. These results open new perspective on the therapeutic potential of this molecule which could have significant beneficial effects on patients with MS and beyond

Philosophiae Doctor - PhD
Tuberculosis (TB) is an infectious disease that, despite all efforts devoted towards its eradication, remains a threat to many countries including South Africa. Current diagnostic assays do offer better performance than the conventional sputum smear microscopy and tuberculin skin tests. However, these assays have been proven to be affected by various factors including the condition of an individual's immune system and vaccination history. By far, electrochemical biosensors are amongst the currently investigated techniques to address the shortcomings associated with these diagnostics.
2020-08-31

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Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.
Para testar a atividade biológica do interferon beta (INF-β) no tratamento da esclerose múltipla (EM), é importante que se tenha um modelo animal. Como a infecção pelo vírus da encefalomielite murina de Theiler (do inglês TMEV – Theiler’s Murine Encephalomyelitis Vírus) é capaz de evoluir para uma lesão desmielinizantesimilar a da EM em humanos, este estudo se propõe a estabelecer os parâmetros para avaliar a infecção do TMEV em culturas de células de rim de hamster neonato (do inglês BHK-21– Baby hamster kidney cells). Para tanto foi necessário adaptar a amostra viral TMEV BeAn à cultura BHK-21, estabelecer um ensaio de RT-PCR e padronizar um PCR em tempo real.Também foi construido um vetor plasmidial contendo o gen L* do TMEV para expressãotransitoria em células HEK-293-T e esta construção plasmidial foi utilizada para obtenção de um soro policlonal anti-L* utilizando a metodologia de imunização genética. Como resultados foram obtidos estoques virais de células BHK-21 infectadas pelo TMEV e parte destes estoques foram avaliados quanto à presença de moléculas genômica TMEV, indicativa de replicação viral, por ensaios de RT-PCR e quantificação por PCR em tempo real. Asregiões do genoma do TMEV 3A3B e L* foram aquelas que forneceram melhores resultadosnesta avaliação genômica quantitativa, que deverá ser aplicada para todos os estoques TMEVBHK-21 que foram obtidos. O vetor plasmidial de expressão células HEK-293-T pcDNA4His/Max contendo o gene L* expressou com sucesso transitoriamente esta proteína heteróloga, porém não foi capaz de induzir a formação de anticorpos policlonais anti-L* em coelhos, através da técnica de imunização genética.
In order to evaluate the biologic activity of the therapeutic drug beta interferon to multiple sclerosis, an adequate animal model is necessary.inthis aspect the infection of murine encephalomyelitis virus can evolve to desmielinization that is very similar to human’s multiple sclerosis, the proposal of this study was the establishment of parameters to evaluate the TMEV infection in baby hamster kidney cells-BHK-21. Toward that objective was adapted the TMEV BeAn prototype sample to BHK-21 and also was established a RT-PCR and a real time PCR. Additionally, it was constructed a plasmid vector containing the L* gene of TMEV, for the expression in HEK-293-T cells. This plasmid vector was evaluated in the capacity to produce anti-L* antibodies by genetic immunization. It was possible to obtain stocks of BHK-21 TMEV infected and some of these contents were evaluated as of the quantity of genomic molecules of TMEV as an indicative of viral replication, by RT-PCR and real time PCR. The TMEV genomic regions 3A3B and L*provided best results in the quantitative evaluation. In thefuture, the real time PCR could be used to evaluate all the stocks that were produced. The plasmidial vector pcDNA4His/Max containing the L* gene was able to transiently express the L* protein, but unfortunately, it was not able to induce anti-L* in rabbits.

Flaviviruses are a major cause of disease in humans and animals worldwide. Tick-borne encephalitis virus (TBEV) is the most relevant arthropod-borne flavivirus endemic in Europe and is the etiological agent of tick-borne encephalitis, a potentially fatal infection of the central nervous system. In our recent work we demonstrated that TBEV is able to trigger the stress response of infected cells leading to the formation of stress granules (SG) (Albornoz et al. 2014). We also found that the formation of SG in TBEV infected cells is delayed, following the same delayed kinetic of the IFN response (Miorin et al. 2012). Indeed, while TBEV replication is evident at early time points post infection, SG and IFN-β mRNA become detectable only after 16 hours. Transcriptome analysis of TBEV infected cells showed that, in addition to interferon and interferon stimulated genes, also genes of the unfolded protein response (UPR) were activated. Interestingly, the spliced form of XBP1 and phosphorylation of PERK occurred early during infection (<12h) indicating that the UPR occurs before induction of interferon. We then investigated the role of the UPR as an early cellular response to the infection and as a possible trigger of the interferon response. Interestingly, when cells were infected following treatment with Tunicamycin, a known inducer of the UPR, the IFN response was already active at 8 hours post-infection and the virus titres were significantly decreased. In this condition formation of stress granules was also anticipated with the same kinetic. These data suggest that TBEV is able to evade both the stress and interferon responses and that the UPR may play a critical and unexpected role in the delayed activation of both.

JESUS, Luciano Augusto Oliveira de. Produção de IFN-Y em pacientes com hanseníase e em seus contactantes numa amostra populacional do município de Sobral-Ceará. 2007. 96 f. Dissertação (Mestrado em Microbiologia Médica) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2007.
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Leprosy, which is caused by Mycobacterium leprae, is an illness of ample clinical and immunopathological spectrum. Its clinical manifestations are correlated with distinct immunologic form, varying from a vigorous immune response mediated by cells to M. leprae, with type 1 standard in the tuberculóide polar region, to an absence of specific cellular response to antigens of M. leprae in the lepromatous polar region, with predominance of type 2 response and exacerbations of humoral response. The capacity of whole M. leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) in the IFN-y production was measured in leprosy patients and their household’s contacts, in the city of Sobral, state of Ceará. A total of 30 leprosy patients were used for the study, before start chemotherapy. The paucibacilary leprosy patient group consisted of eight polar indeterminate, ten polar tuberculóide, two borderline tuberculóide, and the multibacilary leprosy patient group consisted of ten lepromatous leprosy and two borderline lepromatous leprosy. The household contacts group consisted of sixty healthy individuals, consanguineous and non consanguineous. The whole M. leprae antigen stimulated IFN-y production in the PBMC of seven borderline tuberculóide/tuberculóide (DT/TT), three indeterminate form, two borderline lepromatous/lepromatous (DV/VV the DT/TT group produced IFN-y levels significantly higher than DV/VV group (Fisher Test, p=0,027). The IFN-y production in the household contacts was observed in 34, 21 consanguineous and 13 non consanguineous. It wasn’t observed significant difference between paucibacilary household’s contacts (DT/TT, indeterminate form) and multibacilary household’s contacts groups in the IFN-y production to whole M. leprae antigen. However, it was observed significant difference in the production of these cytokine between household contacts and DV/VV patients. This study suggests that there wasn’t significative difference in the production IFN-y between non consanguineous and consanguineous subjects of the paucibacilary and multibacilary cases. Moreover, at correlacionated the production in these cytokine in the subjects with the presence of scar, we didn’t observed significative difference too.
A hanseníase, cujo agente etiológico é o Mycobacterium leprae, é doença de amplo espectro clínico e imunopatológico. Suas apresentações clínicas estão correlacionadas com padrões imunológicos distintos, variando de uma vigorosa resposta imune mediada por células ao M. leprae, com padrão tipo 1 no polo tuberculóide, a uma ausência de resposta celular específica aos antígenos do M. leprae no pólo lepromatoso, com predomínio de resposta tipo 2 e exacerbação da resposta humoral. A capacidade do antígeno bruto de M. leprae em estimular células mononucleares do sangue periférico (PBMC) na produção de IFN-y foi avaliada em pacientes com hanseníase e em seus contactantes, do município de Sobral-CE. Um total de 30 casos foi estudado, antes de receberem tratamento poliquimiterápico. O grupo de casos paucibacilares foi constituído por oito com a forma indeterminada, dez com a forma tuberculóide, dois com a forma dimorfo tuberculóide; e o grupo multibacilar foi constituído por dez com a forma virchoviana e dois com a forma dimorfa virchoviana. O grupo de contactantes foi constituído por sessenta indivíduos, sendo 1 consangüíneo e 1 não consangüíneo para cada caso índice. O antígeno bruto de M. leprae estimulou a produção de IFN-y nas PBMC de sete casos dimorfo tuberculóide/tuberculóide (DT/TT), três com a forma indeterminada e dois com a forma dimorfa virchoviana/virchoviana (DV/VV). O grupo DT/TT produziu níveis de IFN-y significantemente maiores que o grupo DV/VV (Teste de Fisher, p=0,027). A produção de IFN-y nos contactantes foi observada em 34 indivíduos, 21 consangüíneos e 13 não consangüíneos. Não foi observada diferença significativa entre os contactantes do grupo paucibacilar (forma indeterminada, DT/TT) e multibacilares (DV/VV). Porém, foi observada diferença significativa na produção desta citocina entre contactantes e casos DV/VV. O estudo sugere que não há diferença significativa na produção de IFN-y entre indivíduos contactantes consangüíneos e não consangüíneos, dos casos paucibacilares e multibacilares. Além disso, ao correlacionarmos a produção desta citocina nos indivíduos com a presença de cicatriz, também não observamos diferença significativa.

Master of Science
Department of Diagnostic Medicine/Pathobiology
Raymond R. R. Rowland
Interferons (IFNs) comprise a group of antiviral cytokines; however, their expression at the porcine maternal-fetal interface and in fetal tissues has not previously been investigated. The purpose of this study was to analyze the expression of type I IFNs and their receptors in maternal and fetal tissues from sows infected with PRRSV. The approach was to use real-time RT-PCR to identify the expression of different subtypes of type I IFN genes. The results show that the constitutive gene expression of some subtypes including IFN-[alpha] and IFN-[alpha][omega] were detected in fetal lymphoid nodes (IFN-[alpha][omega]), placenta (several IFN-[alpha] subtypes and IFN-[omega]5) and particularly, thymus (multiple IFN-[alpha], IFN-[delta] and IFN-[omega]5). The results demonstrate that porcine type I IFNs are differentially expressed at the maternal-fetal interface and in fetal tissues in response to PRRSV infection.

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FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas
Tuberculosis (TB) is a chronic infection caused by Mycobacterium tuberculosis complex and remains a major worldwide public health problem, leading to almost 1.45 million deaths annually. The state of Amazonas has a high rate incidence of TB, about 68.3/100,000 inhabitants in 2012. Only 5 to 10% of infected individuals develop active TB. It has been suggested that host factors determine the immune response to pathogen. Thus, many immunogenetic researches have demonstrated TB associated genes, but in the north region, research in this field is still rare. This fact motivated the investigation of polymorphisms for IFNG, IL12B, CD80 and CD86 genes, which codify proteins for cellular immune response. Furthermore, IFN- concentration and its relation with genotypes found have been verified. A total of 177 patients and 224 controls (159 contacts and 65 non-contacts) were included in this study and DNA sequencing was performed for genes IFNG (SNP +874A/T and microsatellite +875), IL12B (SNPs +1030C/T, +1188A/C and +1254T/G), CD80 (SNPs -454 C/A, -387 T/C, -232 G/A, -79 C/G, -7T/C, +5C/A and an indel polymorphism -557_-561insCATGA) and CD86 (SNPs +1057G/A and +1079G/A). The IFN-y concentration was determined by enzyme-linked immunoassay. At IFNG, the presence of the allele +874A and the allele with 15 CA repeats were associated with susceptibility to pulmonary TB, while the allele +874T and the allele with 12 CA repeats were associated with protection from pulmonary TB. In addition, an association between genotype CC (SNP +1188A/C at IL12B) and increased risk of pulmonary TB was found. Furthermore, a significant difference between IFN- concentration and genotypes of SNP +1188A/C at IL12B and microsatellite at IFNG was observed, with decrease of IFN-at genotype CC and 15 CA repeats respectively. These outcomes lead to a better understanding of the immune response regulation for TB and help to determine the genetic profile of the Amazon population. Future researches are still needed for a better understanding of the role of other genes involved in the immune response to M. tuberculosis and their influence at the production of citokines like IFN-.
A Tuberculose (TB) é uma infecção crônica causada pelo complexo Mycobacterium tuberculosis sendo um importante problema de saúde pública mundial, levando a aproximadamente 1,45 milhões de mortes a cada ano. O estado do Amazonas possui uma alta incidência desta doença, atingindo 68,3 casos por 100 mil habitantes em 2012. Dos indivíduos infectados pelo bacilo, cerca de 5 a 10% desenvolvem a Tuberculose ativa, sugerindo que há fatores associados ao hospedeiro que determinam o destino da resposta imune ao patógeno. Neste contexto, diversos estudos em imunogenética têm demonstrado genes associados à TB, mas na região norte ainda são raras as pesquisas nesta área, fato que motivou a investigação da frequência dos polimorfismos nos genes IFNG, IL12B, CD80 e CD86, que codificam para proteínas fundamentais na resposta imune celular. Além disso, foi verificado se a concentração de IFN- está relacionada com o genótipo encontrado. Foram incluídas amostras de 177 pacientes e 224 controles (159 contatos e 65 não contatos) e realizado sequenciamento de DNA para os genes IFNG (SNP +874A/T e microssatélite +875), IL12B (SNPs +1030C/T, +1188A/C e +1254T/G), CD80 (SNPs -454 C/A, -387 T/C, -232 G/A, -79 C/G, -7T/C e +5C/A e um polimorfismo indel -557_-561insCATGA) e CD86 (SNPs +1057G/A e +1079G/A). A determinação das concentrações de IFN-foi realizada através de ensaio imunoenzimático. Foi verificada uma associação do gene IFNG, entre a presença do alelo +874A e 15 repetições CA, como fator de risco para TB pulmonar assim como a presença do alelo +874T e 12 repetições CA como fatores de proteção contra TB pulmonar. Também foi encontrada uma associação do genótipo CC, do SNP +1188A/C no gene IL12B, como fator de risco ao desenvolvimento da TB pulmonar. Houve diferença significativa na concentração de IFN-entre os genótipos do SNP +1188A/C no gene IL12B e o microssatélite no gene IFNG, com menor produção no genótipo CC e 15 repetições CA respectivamente. Estes resultados contribuem para o melhor entendimento da regulação na resposta imune à TB e auxilia na determinação do perfil genético da população da região Amazônica. Estudos futuros são necessários para uma melhor compreensão do papel de outros genes envolvidos na resposta imunológica a M. tuberculosis e influência nos níveis de produção de citocinas como IFN-.

The induction of type I interferon (IFN) and subsequent activation of interferon stimulated genes (ISGs) represent a first line of defense against viral infection. Typically type I IFN signaling leads to the phosphorylation of the STAT1 and STAT2 transcription factors (TFs) which then form a trimetric complex with IRF-9 and translocate to the nucleus to induce ISG expression. However, the results of this study showed that IFN-mediated upregulation of the ISG Oas1b, the product of which confers resistance to flavivirus induced disease, can be induced in a STAT1-independent manner. Since numerous ISGs have antiviral functions, many viruses have evolved strategies to disrupt the type I IFN-signaling pathway. In cases when STAT1 activation is blocked by a viral infection, STAT1-independent upregulation of ISGs provides an additional strategy for the cell to mount an effective antiviral response. Infection of mouse embryofibroblasts (MEFs) with West Nile virus (WNV) induced the production of IFN beta and STAT1 and STAT2 phosphorylation but blocked nuclear translocation and binding of these TFs to the promoters of the ISGs, Oas1a, Oas1a, Irf7 and Irf1. However, each of these antiviral ISGs was efficiently upregulated in infected cells and IRF-9 was shown to be crucial for the upregulation of Oas1a, Oas1b and Irf-7. IRF-3 or IRF-7 was needed to maintain the upregulation of these genes at later times of infection. In contrast, the upregulation of Irf1 by WNV infection did not depend on the tested IRFs but was reduced by inhibition of the p38 or NF-kappa B pathways. Although Irf1 mRNA was efficiently upregulated in WNV-infected cells IRF-1 protein synthesis was blocked. The precise mechanism of the IRF-1 translational suppression is not yet known, but the suppression was shown not to be due to increased proteasomal degradation of IRF-1 nor to alternative splicing of Irf1 mRNA. Preliminary results suggest miRNAs may play an indirect role in regulating IRF-1 translation. The results of this study expand knowledge about the strategies evolved by viruses to evade host cell antiviral responses and also provide valuable insights about alternative mechanisms utilized by the host cell to counteract viral infections.

Negli ultimi anni numerose pubblicazioni hanno evidenziato l’influenza che l’IFN di tipo I esercita sul differenziamento, sulla sopravvivenza e sulla funzionalità delle cellule dendritiche e dei linfociti T; in particolare è stato dimostrato l’IFN/ è in grado di indurre in vitro un rapido differenziamento di monociti da sangue periferico in cellule dendritiche particolarmente efficienti nella stimolazione di linfociti T e B (Santini, Lapenta et al. 2000; Honda, Sakaguchi et al. 2003). In questo lavoro abbiamo studiato il ruolo che L’IFN di tipo I riveste sul differenziamento e sulla funzionalità delle cellule dendritiche neonatali. Monociti da sangue di cordone ombelicale sono stati coltivati in vitro in presenza di GM-CSF ed interleuchina 4 (IL4) oppure GM-CSF ed IFN, le cellule dendritiche così ottenute sono state caratterizzate per fenotipo ed attività funzionale. I risultati sono stati paragonati- laddove possibile- con quelli ottenuti dai monociti di sangue periferico. Lo stesso protocollo di coltura in vitro con IFN è stato applicato ai precursori midollari delle cellule dendritiche isolati da topi C57BL6, sempre allo scopo di valutare l’influenza dell’ IFN di tipo I sul loro differenziamento e/o sulla loro maturazione. L’analisi fenotipica delle cellule dendritiche da cordone differenziate con IFN ha evidenziato un fenotipo tipico di cellule immature che esprimono bassi livelli di molecole costimolatorie come CD80 e CD86. Il differenziamento di monociti neonatali in presenza di IL4 ha evidenziato un maggiore grado di immaturità rispetto alla controparte adulta, soprattutto legato ad una espressione ridotta di CD40,CD80 ed HLA-DR. Questi dati sono in accordo con la letteratura corrente, sebbene sia da evidenziare che nel caso di monociti neonatali l’IFN promuove il differenziamento in dendritiche in misura maggiore rispetto al protocollo classico con IL4.D’altra parte monociti provenienti da periferico differenziati in presenza di IFN mostrano un fenotipo semi maturo caratterizzato da una espressione di CD40, CD80, CD86 ed HLA-DR superiore rispetto ai monociti da periferico differenziati in presenza di IL4. I monociti da sangue di cordone risultano essere meno sensibili all’azione dell’IFN rispetto ai monociti da periferico. L’analisi del livello di espressione delle due catene del recettore per l’IFN, IFNAR1 ed IFNAR2, ha rivelato che esiste una modulazione dell’espressione sulla superficie cellulare durante il differenziamento in vitro. Questa modulazione è del tutto sovrapponibile nei monociti da cordone e in quelli da periferico. Allo stesso modo il raffronto fra la percentuale di STAT1 fosforilata rispetto a STAT1, nei monociti neonatali ed adulti , in seguito a stimolazione con IFN, ha dato risultati simili, che si attestano tra valori del 75% e 90% senza evidenziare alcuna differenza per quanto riguarda l’attivazione delle prime fasi del signalling fra cellule adulte e neonatali. Poiché le cellule dendritiche nella loro forma immatura sono in grado di riconoscere gli antigeni mediante recettori di superficie chiamati toll-like receptors, la presenza di questi recettori è stata studiata nei monociti da cordone e da sangue periferico. I monociti da periferico e da cordone esprimono entrambi i TLR2 e TLR4 ma non il TLR3. Inoltre i monociti da cordone esprimono il TLR4 in misura inferiore. Il TLR2 è il recettore per i peptidoglicani mentre il TLR4 è il recettore per il lipopolisaccaride (LPS), entrambi di origine batterica. Da un punto di vista strettamente funzionale i monociti e le dendritiche da cordone non rispondono alla stimolazione in vitro con LPS, che invece costituisce un potente stimolo maturativo insieme al CD40L per monociti e dendritiche da periferico. La capacità delle dendritiche neonatali differenziate con IFN di indurre la proliferazione di linfociti CD4+ e CD8+ da sangue periferico adulto è inferiore rispetto a quella delle dendritiche da cordone differenziate con il protocollo classico. Al contrario dendritiche ottenute in presenza di IFN a partire da cellule del periferico hanno una maggiore capacita allostimolatoria rispetto a quelle derivate in vitro con IL4. Cellule dendritiche da periferico differenziate con entrambi i protocolli mostrano di possedere le stesse attività di endocitosi e macropinocitosi come dimostrato da analisi mediante citofluorimetria dell’uptake con l’utilizzo di antigeni marcati. Per contro dendritiche da cordone differenziate con IFN risultamo meno efficienti nell’endocitare l’antigene rispetto alle dendritiche da cordone differenziate con IL4. Nel modello murino i precursori midollari delle dendritiche di topo sono stati coltivati in vitro in presenza diGMCSF e poi stimolati con IFN, LPS ed HBHA. Quest’ultimo antigene è una emoagglutinina responsabile della disseminazione extrapolmonare del bacillo della tubercolosi. In questo caso l’IFN non si è dimostrato superiore agli altri stimoli nel promuovere la maturazione dei precursori midollari in dendritiche , ma ha dimostrato di avere un effetto sinergico se somministrato in combinazione con l’HBHA, aumentando sensibilmente l’espressione della classe I sulla superficie cellulare. In compenso, in saggi funzionali, le cellule dendritiche stimolate con IFN sono state i più efficaci induttori di proliferazione di splenociti di topo CD4+ e CD8+. E’ stato recentemente dimostrato che un trattamento acuto con l’IFN di classe I è capace di promuovere la proliferazione in vivo di progenitori ematopoietici staminali , mentre la somministrazione continuativa di IFN inibisce i processi di rigenerazione del pool dei progenitori stessi. Si può dunque ipotizzare che l’IFN possa sortire effetti diversi sulle cellule target in funzione del dosaggio e del tipo di somministrazione che viene utilizzato. Pertanto ulteriori studi sono necessari al fine di determinare in modo più dettagliato e conclusivo quale sia l’effetto di questa citochina nel delicato equilibrio che regola la risposta immune nei neonati.
In recent years, a number of reports provided evidence on the importance of type I IFNs in the differentiation, survival and function of several cell types including T cells and dendritic cells (DCs) (Brassard, Grace et al. 2002). IFN-/ has been shown to promote the rapid differentiation of GM-CSF-treated human peripheral blood monocytes into DCs with potent T cell and B cell stimulatory activities (Santini, Lapenta et al. 2000; Honda, Sakaguchi et al. 2003). These results have led to the suggestion that IFN-I could also play a role in the maturation/differentiation of neonatal DCs and in the modulation of Th-1 response which is impaired in neonates. Aim. In this study we focused on DC from umbilical cord blood, as these are the coordinators of the innate as well as the specific immune response, and on the role of type I IFN in their in vitro differentiation/maturation starting from neonatal monocytes. In particular cord blood-derived monocytic cells were cultured in vitro in the presence of GM-CSF and IL-4 or, alternatively, GM-CSF and IFN-α. DCs obtained by the two different methods were analysed in terms of phenotype and functional activity. Wherever possible results from neonatal cord blood-derived DCs were compared with adult peripheral blood-derived DCs. In the mouse model, namely C57BL6 mice, we studied the role of IFN α – in the in vitro differentiation and maturation of bone marrow DC precursors from neonatal mice. Results. Cord blood DCs differentiated in vitro in the presence of IFN were found to have an immature phenotype as they express low levels of costimulatory molecules such as CD80 and CD86. Similar observations were made when neonatal monocytes were cultured in the presence of GM-CSF and IL4 as they displayed reduced HLADR, CD80 and CD40 expression compared to the adult counterparts. Of note, IFN induced differentiation in neonatal cells was still superior to IL4 induced differentiation. No significant differences between cord blood and peripheral blood monocytes were found in the IFN receptor expression during their in vitro differentiation into DCs. The analysis of STAT1, which is the most proximal target of the IFN receptor-associated tyrosine kinases, revealed that a marked tyrosine phosphorylation of STAT1 was observed both in neonatal and adult monocytes at a comparable level ranging from 75% to 90%. DCs in their immature form recognise conserved molecular motifs found in microrganisms by means of pattern-recognition receptors called toll-like receptors (TLRs). The expression pattern of TLRs in IFN-DCs and IL4-DCs have been compared. All monocytes from both peripheral blood (PB) and cord blood (CB) samples expressed TLR-2 and TLR-4 but not TLR-3. Despite the comparable frequencies of monocytes expressing TLR-4, those from cord blood expressed lower amounts of TLR-4 than those from peripheral blood (p < 0.05). From a functional point of view cord blood untreated monocytes and IFN treated DCs showed a deficiency in their ability to respond to LPS and T cell proliferation responses induced by this cells were lower with respect to the peripheral blood counterparts. .Moreover while the majority of PBDCs , either differentiated by IL4 or by IFN, retained comparable phagocytic and processing activity, cord blood IFN-DCs showed a less efficient antigen uptake with respect to the CB IL4-DCs in terms of mannose receptor-mediated endocytosis. Mouse bone marrow derived DCs precursors were coltured in vitro with IFNα alone or in combination with the HBHA (heparin binding haemoagglutinin, a mycobacterial adhesin) antigen and LPS. IFNα treatment proved to be similar to HBHA and LPS stimulation in terms of CD40, CD86 and Class II expression, only the combination of HBHA plus IFNα enhanced DC maturation expecially for Class I expression. The analysis of percentage of mature cells after the stimulation confirmed that IFN alone was not superior to the other stimuli, but it appeared that neonatal bone marrow DCs treated with IFNα, were more efficient inducers of CD4+ and CD8+ mouse lymphocytes proliferation. Conclusions. It is conceivable that IFN can exert opposite effects depending on the timing and the cells differentiation stage, for example by selectively enhancing functional activity at the stage of maturing DCs but exerting an inhibitory effect on DC functions when given at early stages of differentiation. In particular it has been demonstrated that short term IFN stimulation promotes the proliferation of dormant haematopoietic stem cells in vivo whereas chronic IFN treatment exert inhibitory effects on HSC self renewal. Further studies are needed to understand the differential roles that Type I IFN may play in affecting DC function and shaping the immune response in the neonatal environment.