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Ultraviolet radiation (UVR) stimulates melanogenesis in melanocytes, primarily via release of alpha-melanocyte stimulating hormone from keratinocytes. UVR also induced an inflammatory response in the skin in which Interferon-gamma (IFNγ) cytokine plays an important orchestrating role. Here we report that recombinant IFNγ induces a temporal increase of melanogenesis in mouse melanoma cells. IFNγ elevates expression of microphthalmia-associated transcription factor (Mitf), which is the master regulator of melanogenesis by initiating transcription of melanogenic enzymes, tyrosinase (Tyr), tyrosinese-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct). Interestingly, tyrosinase protein, but not mRNA expression, accumulated in response to IFNγ treatment and was consistent with tyrosinase activity. In addition, glycosidase digestion showed that IFNγ induced ER-resistant, fully mature tyrosinase via post-transcriptional mechanisms, rather than increased de novo synthesis or early processing in the ER. Most strikingly, IFNγ mediated alkalization of melanosomes by elevating Oca2 expression, which leads to facilitate melanosome maturation and sequential accumulation of mature tyrosianse. Both Jak1/Jak2 inhibitor Ruxolitinib and knockout of Stat1 mediated by CRISPR-CAS9 blocked the IFNγ-induced Mitf, tyrosinase, Oca2 expressions and melanin biosynthesis. Our data reveals that IFNγ-Jak1/2-Stat1 axis regulates melanogenesis by inducing maturation of melanosomes and accumulation of mature tyrosinase via post-translational mechanisms. CTLA4 is a cell surface receptor on T cells that functions as an immune checkpoint molecule to enforce tolerance to cognate antigens. Anti-CTLA4 immunotherapy is highly effective at reactivating T cell responses against melanoma, which is postulated to be due to targeting CTLA4 on T cells. Here we report that CTLA4 is also highly expressed by most human melanoma cell lines, as well as in normal human melanocytes. Interferon-gamma (IFNγ) signaling activated the expression of the human CTLA4 gene in a melanocyte and melanoma cell-specific manner. Mechanistically, IFNγ activated CTLA4 expression through JAK1/2-dependent phosphorylation of STAT1, which bound a specific gamma-activated sequence (GAS) site on the CTLA4 promoter, thereby licensing CBP/p300-mediated histone acetylation and local chromatin opening. In melanoma cell lines, elevated baseline expression relied upon constitutive activation of the MAPK pathway. Notably, RNA-seq analyses of melanoma specimens obtained from patients who had received anti-CTLA4 immunotherapy (ipilimumab) showed upregulation of an IFNγ -response gene expression signature, including CTLA4 itself, which correlated significantly with durable response. We also show that ectopic expression of Ctla4 in mouse melanoma cells promotes tumor growth in immunocompetent mice. Ctla4-enhanced melanomagenesis is blocked in immunodeficient NSG mice. In addition, ligation of CD86 (one of Ctla4 ligands) in T cells inhibits CD8 T cells proliferation in vitro. Expression of Ctla4 in melanoma cells are resistant to CD8 T cell cytoxicity in vitro. Our data demonstrates and highlights the novel and unrecognized functions of CTLA4 in melanoma cells that aids their survival, immunoevasion and tumorigenic capabilities. Taken together, these findings have potential implications for the conventional and prototypical roles of the IFNγ signaling pathway and CTLA4 in tumor immunosurveillance and tumor immunoevasion. More importantly, our results raise the possibility that CTLA4 targeting on melanoma cells may contribute to the clinical immunobiology of anti-CTLA4 responses. / Biomedical Sciences

Ein Schlüsselprozess in der Immunantwort ist die durch das proinflammatorische Zytokin Interferon-gamma (IFNg) induzierte transiente Akkumulation von neu synthetisierten defekten Proteinen, die durch Anknüpfen von Polymeren des Proteins Ubiquitin (Ub) post-translational modifiziert werden. Die Ubiquitinierung ist das Schlüsselsignal für den Abbau dieser Proteine. Die Abbauprodukte dienen unter anderem als Quelle für die Prozessierung von Antigenen. Um die frühe Immunantwort besser zu verstehen, wurden im Rahmen dieser Arbeit die Identität und Charakteristika dieser neu synthetisierten Proteine, sowie die Topologie ihrer post-translationalen Modifizierung durch Ub untersucht. Dazu wurde die massenspektrometrischen Analyse ubiquitinierter Proteine weiterentwickelt, indem die experimentellen Konditionen auf deren Analyse optimiert wurden. Insbesondere konnte gezeigt werden, dass die kombinierte Verdauung der Ub-Konjugate mittels zweier Peptidasen die Identifizierung der Proteinpeptide und der Indikatorpeptide für Ubiquitinierungsstellen entscheidend verbessert. Es wurde demonstriert, dass eine selektive Isotopenmarkierung der neu synthetisierten Proteine möglich ist. Mit Hilfe dieser Methode gelang es, Veränderungen der Ub Modifikationen bezüglich der Topologie sowie quantitative Unterschiede der ubiquitinierten Proteine aus humanen HeLa-Zellen in An- und Abwesenheit von IFNg zu identifizieren. Nach Induktion durch IFNg wurden drei polyubiquitinierte, drei mono- oder polyubiquitinierte und 111 potentiell ubiquitinierte Proteine identifiziert. Diese Proteine zeigten, dass keine generelle Ubiquitinierungspräferenz für die durch IFNg verstärkt transkribierten Gene besteht. Die Ergebnisse dieser Arbeit erweitern das Verständnis der frühen zellulären Immunantwort und tragen zum Verständnis von Krebs- und Autoimmunerkrankungen sowie chronischen Entzündungsprozessen bei. Möglicherweise bilden sie die Grundlage für die Weiterentwicklung entsprechender Therapien. / A key process within the immune response of organisms is the transient accumulation of newly synthesized defective proteins affected by the proinflammatory cytokine interferon-gamma(IFNg). These proteins are post-translationally modified by attachment of polymers of the protein ubiquitin (Ub), which represents the key signal for targeting them to degradation. The resulting peptides can serve as sources for antigen processing. In order to get an insight into the details of the cellular early immune response, the identity and characteristics of the newly synthesized proteins and the topology of their post-translational modification with Ub were investigated. An essential progress of the mass spectrometric approach was achieved by optimizing the experimental conditions with regard to the requirements of the analysis of the targeted proteins. In particular, it could be shown that a combined digestion of Ub-conjugates with two peptidases leads to an improved detection of protein peptides and indicator peptides for ubiquitination sites. It was shown that a selective isotopic labeling of the newly synthesized proteins was possible. With this method, a decisive step forward was made in the understanding of changes of the Ub modifications with respect to the topology and in clarifying the quantitative differences between Ub-conjugates from IFNg treated and untreated human HeLa cells. Three IFNg induced polyubiquitinated proteins, three mono- or polyubiquitinated as well as 111 potential Ub-substrates could be identified. These proteins did not show any general ubiquitination preference for genes whose transcription is enhanced in presence of IFNg. The results obtained in this work help to broaden and refine the general picture of the early cellular immune response. They contribute to the knowledge on molecular processes of cancer, autoimmune diseases or chronic inflammation and, potentially, can give hints for the continued development of corresponding therapies.

Das Oberflächenepithel (OSE) des Ovars besteht aus einer Schicht flacher oder kubischen Zellen, die am Hilus in das flache Peritonealepithel übergehen. Das OSE zeigt zyklusabhängige Veränderungen. OSE-Zellen sollen am Prozess der Ovulation aktiv teilnehmen und die Läsion nach der Follikelruptur reparieren. Die mitotische Aktivität der OSE-Zellen um den Reparatur-Prozess könnte das Überleben von mutierten Zellen begünstigen und zum Ovarialkarzinom führen. Hierbei spielen Zell-Zell-Kontakte eine Schlüsselrolle bei der Integrität von Gewebe und der Metastasierung von Tumoren. Um das Verständnis über die Pathogenese des Ovarialkarzinoms zu verbessern, untersuchte die vorliegende Arbeit die Zell-Zell-Kontakte des OSE sowie dessen Abhängigkeit von Interferon-γ (IFN-γ), welches u.a. bei der adjuvanten Therapie des Ovarialkarzinoms verwendet wird. Abstriche von humanen und bovinen Ovarien dienten als Quelle zur Kultivierung von OSE-Zellen. Konfluente Kulturen wurden mit 200 U/ml rekombinantem, speziesspezifischem IFN-γ für 72 h behandelt oder als Kontrolle unbehandelt gelassen. Die Morphologie der OSE-Zellen vor und nach der Behandlung wurde dargestellt. Weiterhin wurden mittels immunzytologischer Färbungen sowie Western Blot Analyse E- und N-Cadherin, β-Catenin, Cytokeratin sowie Vimentin nachgewiesen. Permeabilitätsmessungen von Meerrettichperoxidase (HRP) in einem Ko-Kultursystem wurden mit und ohne IFN-γ durchgeführt. Die Arbeit zeigt eine neue Wirkung von IFN-γ. Es hat die besondere Eigenschaft, das OSE komplett über Cadherin-vermittelte „tight juctions“ abzudichten. Dies könnte die Wirksamkeit des Zytokins bei der adjuvanten Therapie des Ovarialkarzinoms erklären. Möglicherweise werden Interzellularkontakte verstärkt und die Frühinvasion maligner Zellen eingeschränkt. Das erstmals beschriebene Cadherinmuster an den Zell-zu-Zell-Ecken verweist auf die Interaktion der „tight junctions“ mit E-Cadherin.

O vírus linfotrópico de células T humanas do tipo 1 (HTLV-1) é o agente etiológico de duas principais manifestações clínicas, a mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP) e a leucemia/linfoma de células T do adulto (ATLL). O desenvolvimento de HAM/TSP, uma doença neuroinflamatória crônica é relacionada a uma complexa interação entre vírus e resposta imunológica do hospedeiro. A expressão do gene viral tax desempenha um papel importante na patogênese da HAM/TSP pela ativação de diversos genes celulares, incluindo genes das citocinas inflamatórias IFN-? e TNF- Recentemente, os exossomos surgiram como um importante fator na comunicação célula-célula contribuindo em diversos processos celulares, inclusive na modulação do sistema imunológico. Considerando o potencial papel dos exossomos na imunomodulação e no processo inflamatório, o objetivo deste trabalho foi avaliar se os exossomos produzidos por células infectadas pelo HTLV-1 carreiam moléculas virais e se poderiam participar dos processos de imunomodulação e inflamação crônica observados na HAM/TSP. Os exossomos foram isolados de células infectadas pelo HTLV-1 da linhagem celular C8166, avaliados quanto à presença de RNAm viral tax e testados quanto à capacidade de ativar células mononucleares do sangue periférico (PBMC) à indução de resposta inflamatória. Neste trabalho, foi observado que, após a exposição de PBMC aos exossomos contendo RNAm viral tax, a expressão da citocina inflamatória IFN-? foi aumentada em linfócitos T CD4+ e CD8+ quando comparado ao grupo não exposto aos exossomos. Foi observado um pequeno aumento de TNF-? no grupo exposto aos exossomos. Não houve diferença na expressão das citocinas IL-4, perforina e granzima B. Tomados em conjunto, estes resultados sugerem que exossomos contendo tax isolados de células infectadas pelo HTLV-1 induzem a produção de citocinas inflamatórias ativam a resposta imunológica de padrão Th1, e não Th2. Estes achados podem contribuir na elucidação do papel dos exossomos na infecção pelo HTLV-1 e nos estudos relacionados à patogênese da HAM/TSP e à resposta inflamatória envolvendo a apresentação clínica desta doença. / Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of HTLV-1 associated myelopathy/tropical paraparesis spastic (HAM/TSP) and adult T-cell leukemia/lymphoma (ATLL). The development of HAM/TSP, a chronic neuroinflammatory disease, is correlated to a complex host-virus dynamic immune response interaction. Tax expression plays an important role in HAM/TSP pathogenesis by activating various cellular genes, including the cytokines IFN-? and TNF-?. Exosomes have emerged as an important factor of cell-to-cell communication contributing to diverse cellular processes, including immune modulation. Considering the potential role of exosomes in modulating the immune response and inflammation, the main objective of this study was to examine if HTLV-1-infected cells produce exosomes can carry viral tax mRNA, and if they can can participate in the chronic inflammation observed in patients with HAM/TSP. Exosomes were isolated from HTLV-1-infected cell line culture, evaluated for the tax mRNA presence and tested for the ability to activate peripheral mononuclear cells (PBMC) in inducing an inflammatory immune response. We observed that the proinflammatory cytokine IFN-? was upregulated in CD4 and CD8 T-cells after treatment of the PBMC with Tax carrying exosomes compared to the negative control. The expression of TNF-? was slightly upregulated. IL-4, Granzyme B and Perforin did not show alterations. Taken together, these results suggest that exosomes carrying Tax isolated from HTLV-1-infected cells might induce the production of proinflammatory cytokines and activate T helper (Th)1, and not Th2, immune response. This study may have important impact on the studies involving the pathogenesis of HAM-TSP and the inflammatory response involved in the clinical presentation of the disease.

Mutações no gene CD40LG estão associadas à Síndrome de Hiper-IgM, relacionada ao cromossomo X, que é uma Imunodeficiência Primária. A ausência da interação CD40L/CD40 (linfócitos-DCs) acarreta um aumento da suscetibilidade às infecções fúngicas. Os efeitos do tratamento in vitro com interferon-gama em células dendríticas de pacientes com mutações no CD40L demonstraram potencial sobre a imunologia das DCs e nos remete a fundamentar possíveis ensaios clínicos futuros, que tenham por tema a prevenção e o tratamento de infecções oportunistas. / Mutations in the CD40LG gene are associated with the X-linked Hyper-IgM Syndrome, which is a Primary Immunodeficiency. The absence of the CD40L / CD40 interaction (lymphocytes-DCs) leads to increased susceptibility to fungal infections. The effects of in vitro gamma-interferon treatment on dendritic cells from patients with CD40L mutations have demonstrated potential for DC immunology and suggest that future clinical trials should address the prevention and treatment of opportunistic infections.

A hepatite C é uma doença que leva à inflamação do fígado, sendo causada pelo vírus da hepatite C (VHC). Estima-se que existam 130 a 170 milhões de casos de infecção crônica pelo VHC por todo o mundo. Apesar do VHC ser principalmente hepatotrópico, foi evidenciada a detecção de RNA viral em células mononucleares do sangue periférico (PBMC). Evidências clínicas e experimentais têm demonstrado um importante tropismo do VHC por células do sistema imune, em especial por PBMCs. Apesar deste interessante achado, a importância da infecção do sistema imune na história natural da doença não é totalmente conhecida. É possível observar em alguns estudos que, em pacientes que desenvolvem infecção aguda, observa-se uma vigorosa resposta mediada por células T, específica ao VHC, mediada, por células T CD4+ e T CD8. Esta resposta é detectada na fase inicial da doença, e prolonga-se durante vários anos após a resolução do vírus. Pelo contrário, os pacientes que desenvolvem infecção crônica, apresentam, normalmente, respostas T fracas e / ou de curta duração, bem como defeitos nas funções efetoras de células T específicas. As respostas mediadas por células T com este perfil resultam, normalmente, em um baixo controlo da viremia e na sua persistência. Outra importante questão que permanece incerta até o presente momento é como a infecção das células imunes pelo VHC altera a sua função, principalmente no que concerne às PBMCs. Informações referentes às PBMCs e o VHC são pouco estudadas e referidas na literatura internacional, embora sejam de fundamental importância para a compreensão do seu peso na história natural da infecção. OBJETIVO: Baseado nessas incertezas o presente estudo tentará contribuir para a elucidação da influência do parasitismo por VHC na função de células TCD4+ e TCD8+ em pacientes naïve com indicação de tratamento com IFNpeg+RBV e comparar os dados obtidos antes, durante e pós-tratamento com os valores encontrados dos controles negativos, avaliando a influência do tratamento sobre a função dessas células. Verificar a presença do RNA-VHC nas PBMCs por RT-PCR em Tempo Real. Verificar a influência de fatores do vírus e do hospedeiro, tais como genótipo, carga viral, idade, gênero, raça, polimorfismo IL28B e biópsia hepática sobre as células parasitadas pelo VHC e sua produção de citocinas; Determinar a contagem e a função de linfócitos T CD4+ e CD8+ no sangue periférico desses pacientes; Determinar a influência do parasitismo de linfócitos pelo VHC sobre a função dos linfócitos TCD4+ e TCD8+. METODOLOGIA: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios, atendidos no Ambulatório de Hepatites da Divisão de Clínica de Doenças Infecciosas e Parasitárias do HC FMUSP. A contagem de células da subpopulação de linfócitos T CD4+ e CD8+ periféricas foram realizadas por citometria de fluxo FACSCanto II (BD) por meio do software MultiSet(BD). Foi analisada a presença/ausência do RNA-VHC em PBMC por PCR em tempo real no sistema TaqMan, no termociclador Applied Biosystems StepOne(TM). A função dos linfócitos TCD4+ e TCD8+ foram avaliadas através da técnica ELISPOT utilizanso o Kit Human IFN-gamma/IL-4 Dual-Color FluoroSpot. A contagem dos spots foi realizada em um leitor automatizado CTL-ImmunoSpot® S6 FluoroSpot Line. As PBMCs foram fracionadas e depletadas, utilizando-se o Kit Dynabeads FlowComp Human CD4 (Invitrogen Life Technologies) segundo as instruções do fabricante. RESULTADOS: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios. O genótipo 1 foi o mais prevalente 61,5%. O RNA do VHC foi detectado nas PBMCS em 88,4% dos pacientes. XVII Nossos resultados mostraram um aumento de células CD4 e CD8 parasitadas pelo VHC antes do tratamento, com valores estatisticamente significantes quando comparadas aos controles normais apenas no caso das CD4. Ocorreu, porém, um nítido prejuízo na produção de interleucinas por estas células parasitadas, particularmente a produção de IFN- y, com valores altamente significantes (0,009). Na semana 12, podemos ver o aumento de células CD4 no pré-tratamento, porém com diminuição na semana 12 e no follow up; entretanto a produção de IL-4 pelas células CD4 aumenta na semana 12 e cai novamente no seguimento; com as células CD8 ocorre leve queda na semana 12 porém uma tentativa de recuperação no follow up; sua produção de IFN-y cai na semana 12 e no follow up para números estatisticamente significantes. É a chamada \"exaustão\" de função destas células já descrita in vitro por alguns autores. Estes dados poderão ser muito úteis nas futuras observações desses pacientes que serão tratados com esquemas de DAAs sem interferon alfa. CONCLUSÃO: Em conclusão, os resultados desta pesquisa confirmam a importante influência do parasitismo das células CD4 e CD8 em suas funções / Hepatitis C is a disease that causes inflammation of the liver, caused by hepatitis C virus (HCV). It is estimated that there are 130 million to 170 million cases of chronic HCV infection worldwide. Although HCV is primarily hepatotropic was evidenced by detection of viral RNA in peripheral blood mononuclear cells (PBMC). Clinical and experimental evidence have demonstrated an important tropism of HCV by immune system cells, in particular by PBMC. Despite this interesting finding, the importance of infection of the immune system in the natural history of the disease is not fully known. It can be observed in some studies in patients who develop acute infection a vigorous response is observed mediated by T cells specific to HCV mediated by CD4 + and CD8. This response is detected at the initial stage of the disease, and extends for years after the resolution of the virus. Conversely, patients who develop chronic infection, are typically in the low T responses and / or short-term as well as defects in the effector functions of specific T cells. The responses mediated by T cells with the profile usually result in a low control of viremia and its persistence. Another important issue that remains unclear to date is how the infection of immune cells by HCV alters its function, especially with regard to the PBMCs. Information relating to the PBMCs and HCV are little studied and reported in the international literature, although they are of fundamental importance for the understanding of its weight in the natural history of infection. OBJECTIVE: Based on these uncertainties the present study attempts to contribute to the elucidation of the influence of parasitism by HCV on CD4 T cell function + and CD8 + in naïve patients with treatment indication with IFNpeg + RBV and compare the data obtained before, during and after treatment with the values found negative controls, assessing the influence of treatment on the function of these cells. Verify the presence of HCV-RNA in PBMC by RT-PCR in real time. Check the influence of virus and host factors such as genotype, viral load, age, gender, race, IL28B polymorphism and liver biopsy on the parasitized cells with HCV and their cytokine production; Determine count and function of lymphocytes T CD4 + and CD8 + peripheral blood of patients; To determine the influence of parasitism lymphocytes HCV on the function of CD4 + and CD8 + T lymphocytes. METHODS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls, treated at the Hepatitis Clinic of the Division of Clinical Infectious and Parasitic Diseases of the FMUSP. The lymphocyte subpopulation of T-cell count CD4 + and CD8 + peripheral been made by FACSCanto II flow cytometer (BD) through the multiset software (BD). the presence / absence of HCV-RNA in PBMC by real-time PCR was analyzed in the TaqMan system, Applied Biosystems thermal cycler StepOne (TM). The role of CD4 + T lymphocytes and CD8 + were assessed by ELISPOT technique utilizanso the Human Kit IFN-gamma / IL-4 Dual-Color FluoroSpot. The counting of the spots was carried out in an automated reader CTL-ImmunoSpot® S6 FluoroSpot Line. And fractionated PBMCs were depleted using Dynabeads kit is the Human CD4 FlowComp (Invitrogen Life Technologies) according to manufacturer\'s instructions. RESULTS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls. Genotype 1 was the most prevalent 61.5%. The HCV RNA was detected in PBMC in 88.4% of patients. Our results showed an increase of CD4 and CD8 cells parasitized with HCV prior to treatment, with statistically significant amounts compared to controls only if CD4. There was however, a marked impairment in production of interleukins for these parasitized cells, particularly the production of IFN- y, with highly significant values (0.009). At week 12, we can see the increase in CD4 cells before treatment, but with decreased at week 12 and at follow up; However IL-4 production by CD4 cells increased at week 12 and again falls in the following; with the CD8 cells is slightly lower at week 12 but a recovery attempt at follow up; their IFN-y production drops at week 12 and follow up to statistically significant numbers. It is called \"exhaust\" function of these cells in vitro been described by some authors. This data is very useful in future observations of these patients are treated with AADs regimens without interferon alfa. CONCLUSION: In conclusion, the results confirm the important influence of the parasitism of CD4 and CD8 cells in their functions

A hepatite C é uma doença que leva à inflamação do fígado, sendo causada pelo vírus da hepatite C (VHC). Estima-se que existam 130 a 170 milhões de casos de infecção crônica pelo VHC por todo o mundo. Apesar do VHC ser principalmente hepatotrópico, foi evidenciada a detecção de RNA viral em células mononucleares do sangue periférico (PBMC). Evidências clínicas e experimentais têm demonstrado um importante tropismo do VHC por células do sistema imune, em especial por PBMCs. Apesar deste interessante achado, a importância da infecção do sistema imune na história natural da doença não é totalmente conhecida. É possível observar em alguns estudos que, em pacientes que desenvolvem infecção aguda, observa-se uma vigorosa resposta mediada por células T, específica ao VHC, mediada, por células T CD4+ e T CD8. Esta resposta é detectada na fase inicial da doença, e prolonga-se durante vários anos após a resolução do vírus. Pelo contrário, os pacientes que desenvolvem infecção crônica, apresentam, normalmente, respostas T fracas e / ou de curta duração, bem como defeitos nas funções efetoras de células T específicas. As respostas mediadas por células T com este perfil resultam, normalmente, em um baixo controlo da viremia e na sua persistência. Outra importante questão que permanece incerta até o presente momento é como a infecção das células imunes pelo VHC altera a sua função, principalmente no que concerne às PBMCs. Informações referentes às PBMCs e o VHC são pouco estudadas e referidas na literatura internacional, embora sejam de fundamental importância para a compreensão do seu peso na história natural da infecção. OBJETIVO: Baseado nessas incertezas o presente estudo tentará contribuir para a elucidação da influência do parasitismo por VHC na função de células TCD4+ e TCD8+ em pacientes naïve com indicação de tratamento com IFNpeg+RBV e comparar os dados obtidos antes, durante e pós-tratamento com os valores encontrados dos controles negativos, avaliando a influência do tratamento sobre a função dessas células. Verificar a presença do RNA-VHC nas PBMCs por RT-PCR em Tempo Real. Verificar a influência de fatores do vírus e do hospedeiro, tais como genótipo, carga viral, idade, gênero, raça, polimorfismo IL28B e biópsia hepática sobre as células parasitadas pelo VHC e sua produção de citocinas; Determinar a contagem e a função de linfócitos T CD4+ e CD8+ no sangue periférico desses pacientes; Determinar a influência do parasitismo de linfócitos pelo VHC sobre a função dos linfócitos TCD4+ e TCD8+. METODOLOGIA: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios, atendidos no Ambulatório de Hepatites da Divisão de Clínica de Doenças Infecciosas e Parasitárias do HC FMUSP. A contagem de células da subpopulação de linfócitos T CD4+ e CD8+ periféricas foram realizadas por citometria de fluxo FACSCanto II (BD) por meio do software MultiSet(BD). Foi analisada a presença/ausência do RNA-VHC em PBMC por PCR em tempo real no sistema TaqMan, no termociclador Applied Biosystems StepOne(TM). A função dos linfócitos TCD4+ e TCD8+ foram avaliadas através da técnica ELISPOT utilizanso o Kit Human IFN-gamma/IL-4 Dual-Color FluoroSpot. A contagem dos spots foi realizada em um leitor automatizado CTL-ImmunoSpot® S6 FluoroSpot Line. As PBMCs foram fracionadas e depletadas, utilizando-se o Kit Dynabeads FlowComp Human CD4 (Invitrogen Life Technologies) segundo as instruções do fabricante. RESULTADOS: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios. O genótipo 1 foi o mais prevalente 61,5%. O RNA do VHC foi detectado nas PBMCS em 88,4% dos pacientes. XVII Nossos resultados mostraram um aumento de células CD4 e CD8 parasitadas pelo VHC antes do tratamento, com valores estatisticamente significantes quando comparadas aos controles normais apenas no caso das CD4. Ocorreu, porém, um nítido prejuízo na produção de interleucinas por estas células parasitadas, particularmente a produção de IFN- y, com valores altamente significantes (0,009). Na semana 12, podemos ver o aumento de células CD4 no pré-tratamento, porém com diminuição na semana 12 e no follow up; entretanto a produção de IL-4 pelas células CD4 aumenta na semana 12 e cai novamente no seguimento; com as células CD8 ocorre leve queda na semana 12 porém uma tentativa de recuperação no follow up; sua produção de IFN-y cai na semana 12 e no follow up para números estatisticamente significantes. É a chamada \"exaustão\" de função destas células já descrita in vitro por alguns autores. Estes dados poderão ser muito úteis nas futuras observações desses pacientes que serão tratados com esquemas de DAAs sem interferon alfa. CONCLUSÃO: Em conclusão, os resultados desta pesquisa confirmam a importante influência do parasitismo das células CD4 e CD8 em suas funções / Hepatitis C is a disease that causes inflammation of the liver, caused by hepatitis C virus (HCV). It is estimated that there are 130 million to 170 million cases of chronic HCV infection worldwide. Although HCV is primarily hepatotropic was evidenced by detection of viral RNA in peripheral blood mononuclear cells (PBMC). Clinical and experimental evidence have demonstrated an important tropism of HCV by immune system cells, in particular by PBMC. Despite this interesting finding, the importance of infection of the immune system in the natural history of the disease is not fully known. It can be observed in some studies in patients who develop acute infection a vigorous response is observed mediated by T cells specific to HCV mediated by CD4 + and CD8. This response is detected at the initial stage of the disease, and extends for years after the resolution of the virus. Conversely, patients who develop chronic infection, are typically in the low T responses and / or short-term as well as defects in the effector functions of specific T cells. The responses mediated by T cells with the profile usually result in a low control of viremia and its persistence. Another important issue that remains unclear to date is how the infection of immune cells by HCV alters its function, especially with regard to the PBMCs. Information relating to the PBMCs and HCV are little studied and reported in the international literature, although they are of fundamental importance for the understanding of its weight in the natural history of infection. OBJECTIVE: Based on these uncertainties the present study attempts to contribute to the elucidation of the influence of parasitism by HCV on CD4 T cell function + and CD8 + in naïve patients with treatment indication with IFNpeg + RBV and compare the data obtained before, during and after treatment with the values found negative controls, assessing the influence of treatment on the function of these cells. Verify the presence of HCV-RNA in PBMC by RT-PCR in real time. Check the influence of virus and host factors such as genotype, viral load, age, gender, race, IL28B polymorphism and liver biopsy on the parasitized cells with HCV and their cytokine production; Determine count and function of lymphocytes T CD4 + and CD8 + peripheral blood of patients; To determine the influence of parasitism lymphocytes HCV on the function of CD4 + and CD8 + T lymphocytes. METHODS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls, treated at the Hepatitis Clinic of the Division of Clinical Infectious and Parasitic Diseases of the FMUSP. The lymphocyte subpopulation of T-cell count CD4 + and CD8 + peripheral been made by FACSCanto II flow cytometer (BD) through the multiset software (BD). the presence / absence of HCV-RNA in PBMC by real-time PCR was analyzed in the TaqMan system, Applied Biosystems thermal cycler StepOne (TM). The role of CD4 + T lymphocytes and CD8 + were assessed by ELISPOT technique utilizanso the Human Kit IFN-gamma / IL-4 Dual-Color FluoroSpot. The counting of the spots was carried out in an automated reader CTL-ImmunoSpot® S6 FluoroSpot Line. And fractionated PBMCs were depleted using Dynabeads kit is the Human CD4 FlowComp (Invitrogen Life Technologies) according to manufacturer\'s instructions. RESULTS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls. Genotype 1 was the most prevalent 61.5%. The HCV RNA was detected in PBMC in 88.4% of patients. Our results showed an increase of CD4 and CD8 cells parasitized with HCV prior to treatment, with statistically significant amounts compared to controls only if CD4. There was however, a marked impairment in production of interleukins for these parasitized cells, particularly the production of IFN- y, with highly significant values (0.009). At week 12, we can see the increase in CD4 cells before treatment, but with decreased at week 12 and at follow up; However IL-4 production by CD4 cells increased at week 12 and again falls in the following; with the CD8 cells is slightly lower at week 12 but a recovery attempt at follow up; their IFN-y production drops at week 12 and follow up to statistically significant numbers. It is called \"exhaust\" function of these cells in vitro been described by some authors. This data is very useful in future observations of these patients are treated with AADs regimens without interferon alfa. CONCLUSION: In conclusion, the results confirm the important influence of the parasitism of CD4 and CD8 cells in their functions

Ebola virus (EBOV) is a member of the Filoviridae family of highly pathogenic viruses that cause severe hemorrhagic fever and is the causative agent of the 2014 West Africa outbreak. Currently, there are no approved filovirus vaccines or treatments to combat these sporadic and deadly epidemics. One target for EBOV antiviral therapy is to block viral entry into host cells. Recently, phosphatidylserine (PtdSer) receptors, primarily known for their involvement in the clearance of dying cells, were shown to mediate entry of enveloped viruses including filoviruses. The PtdSer receptors, T-cell immunoglobulin mucin domain-1 (TIM-1) and family member TIM-4, serve as filovirus receptors, significantly enhancing EBOV entry. TIM-dependent virus uptake occurs via apoptotic mimicry by binding to PtdSer on the surface of virions through a conserved PtdSer binding pocket within the amino terminal IgV domain. TIM-4 is expressed on antigen presenting cells (APCs), including macrophages and dendritic cells (DCs), which are critical in early EBOV infection. My studies are the first to define the molecular details of virion/TIM-4 interactions and establish the importance of TIM-4 for EBOV infection of murine resident peritoneal macrophages. In addition, previous work has utilized only in vitro models to establish the importance of the TIM proteins in EBOV entry. My studies are the first to demonstrate the importance of TIM-1 and TIM-4 for in vivo EBOV pathogenesis and to confirm them as relevant targets of future filovirus therapeutics. Macrophage phenotypes can vary greatly depending upon chemokine and cytokine signals from their microenvironment. Historically, macrophages have been classified into two major subgroups: classically activated macrophages (M1) and alternatively activated macrophages (M2). Macrophages are a critical early target of EBOV infection and my work primarily focused on interferon gamma-stimulated (M1) macrophages since this treatment profoundly inhibited EBOV infection of human and murine macrophages. Interferon gamma treatment blocked EBOV replication in macrophages, reducing viral RNA levels in a manner similar to that observed when cultures were treated with the protein synthesis inhibitor, cycloheximide. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited EBOV infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit infection of negative strand RNA viruses including EBOV. In addition and most exciting, using MA-EBOV, we found that murine interferon gamma, when administered either 24 hours before or after infection, protects lethally challenged mice and significantly reduces morbidity. Our findings suggest that interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option.