Lung cancer is one of the main causes of death worldwide, with most patients suffering from an advanced unresectable or metastatic non-small cell lung cancer. The mortality trends are mostly related to patterns of tobacco use, specifically from cigarettes. Tobacco is the basic etiological agent found as a consequence of the inhalation of tobacco smoke. Published data show the use of interferons (IFNs) in the treatment of lung tumours due to their potential in displaying antiproliferative, anti-angiogenic, immunoregulatory, and proapoptotic effects. Type1 IFNs have been employed as treatments for many types of cancer, both for haematological cancers and solid tumours. The IFN-γ (naked) functions as an anticancer agent against various forms of cancer. Hence, this study aimed to investigate the genoprotective and genotoxic effects of IFN-γ liposome (nano) on 42 blood samples from lung cancer patients, compared to the same sample size from healthy individuals. The effectiveness of IFN- γ liposome against oxidative stress was also evaluated in this study. A concentration of 100U/ml of IFN-γ liposome was used to treat the lymphocytes in: Comet and micronucleus assays, Comet repair, Western blotting and real-time polymerase chain reaction (qPCR) were based on a preliminary test for the optimal dose. The lymphocytes from lung cancer patients presented with higher DNA damage levels than those of healthy individuals. IFN-γ liposome was not found to induce any DNA damage in the lymphocytes. Also, it caused a significant reduction in DNA damage in the lymphocytes from lung cancer patients in; Comet, Comet repair and micronucleus assays. Furthermore, the 100U/ml of IFN-γ liposome significantly reduced the oxidative stress caused by H2O2 and appeared to be effective in both groups using the Comet and micronucleus assays. Results from; Comet, Comet repair and micronucleus assays were consistent. The data obtained indicated that IFN-γ in both forms (naked INF-γ and INF-γ nano-liposome) may potentially be effective for the treatment of lung cancer and showed the ability of IFN-γ liposome to reduce the DNA damage more than the naked form. The IFN-γ in both forms has also shown anti-cancer potential in the lymphocytes from lung cancer patients by regulating the expression of p53, p21, Bcl-2 at mRNA and protein levels by up-regulating the p53 and p21 to mediate cell cycle arrest and DNA repair in lung cancer patients. The findings of this study are consistent with the view that the naked IFN-γ and liposome could have a significant role in cancer treatment, including lung cancer. / Mutah University in Jordan

No description available.

No description available.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24046号 / 生博第472号 / 新制||生||63(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 上村 匡, 教授 井垣 達吏 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Dentre pacientes com imunodeficiências primárias, existem aqueles com defeitos de fagócitos e outros componentes da imunidade inata. A doença granulomatosa crônica (DGC) é uma imunodeficiência primária (IDP) causada por mutações em um dos componentes protéicos, gp91-phox, p22-phox, p47-phox, p67-phox e p40-phox, da nicotinamida adenina dinucleotídeo fosfato (NADPH) dos fagócitos. Pacientes com DGC apresentam maior susceptibilidade a infecções, assim como hiperinflamação e reação adversa à vacinas como à do Bacilo Calmette-Guérin (BCG), como consequência da atividade microbicida defeituosa dos fagócitos. Por outro lado, a susceptibilidade mendeliana a micobactérias (MSMD) é uma condição que predispõe os pacientes a infecções pelo gênero Mycobacterium sp, levando a infecções graves e por vezes à morte. O objetivo deste trabalho foi realizar o diagnóstico clínico e a análise genético-molecular de pacientes brasileiros com DGC e MSMD. A explosão respiratória de granulócitos foi avaliada pelo ensaio de dihidrorodamina (DHR). A dosagem de citocinas do eixo IL-12/IFN-&#947 foi realizada mediante o ensaio de ELISA após estimulo com lisado de micobactérias (LM), proteína purifica (PPD) e BCG. O DNA genômico dos pacientes foi extraído, amplificado e sequenciado pelo método de Sanger e seqüenciamento completo de exoma. Durante o período de 2014-2018, 181 pacientes com histórico clínico sugestivo de DGC e 75 pacientes com diagnóstico sugestivo de MSMD foram encaminhados ao nosso laboratório. Após avaliação clínica e bioquímica dos pacientes, 23 deles foram diagnosticadas com DGC e 16 com MSMD. A análise genético-molecular permitiu identificar mutações em 14 pacientes com DGC, nove deles com DGC ligada ao cromossomo X (DGC-X) e 5 com DGC autossômica recessiva (DGC-AR). Identificamos mutações em 5 pacientes com MSMD, sendo três delas no receptor de IL-12 e duas no receptor da IL-17. / Among patients with primary immunodeficiencies, there are those with defects in phagocytes and other components of the innate immunity. Chronic granulomatous disease (CGD) is a primary immunodeficiency (PID) caused by mutations in one of the protein components, gp91-phox, p22-phox, p47-phox, p67-phox and p40-phox of the Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase of phagocytes. Patients with CDG are susceptible to infections, as well as hyperinflammation and adverse reactions to vaccines such as Bacilo Calmette-Guérin (BCG) as a consequence of defective phagocytes microbicidal activity. On the other hand, Mendelian susceptibility to mycobacterial diseases (MSMD) is a condition that predisposes patients to infections by the genus Mycobacterium sp , leading to serious infections and sometimes death. The main goal of this study was to perform the clinical diagnosis and genetic-molecular analysis of Brazilian patients with CDG and MSMD. The respiratory burst of granulocytes was evaluated by the dihydrorhodamine (DHR) assay. Cytokine dosing of IL-12 / IFN-&#947 axis was performed by the ELISA assay after stimulation with mycobacterium lysate (LM), purified protein (PPD) and BCG. Patients genomic DNA was extracted, amplified and sequenced by the Sanger method and whole exome sequencing. During the period of 2014 to 2018, 181 patients with a clinical history suggestive of CDG and 75 patients with a diagnosis suggestive of MSMD were referred to our laboratory. After clinical and biochemical evaluation, 23 of them were diagnosed with CDG and 16 with MSMD. Genetic-molecular analysis allowed the identification of mutations in 14 patients with CDG, of those 9 had X-linked DGC (X-CGD) and 5 had autosomal recessive CGD (AR-CGD). Mutations were identified in 5 MSMD patients, three in the IL-12 receptor and two in the IL-17 receptor.

All viruses encode for at least one viral interferon (IFN) antagonist, which is used to subvert the cellular IFN response, a powerful antiviral innate immune response. Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial for virus survival, suggesting that viral IFN antagonists could represent promising therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an important human pathogen for which there is no vaccine or virus-specific antiviral drug. RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV replication and pathogenicity. We developed a high-throughput screening (HTS) assay to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN- signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using a HTS approach, we screened 16,000 compounds to identify small molecules that inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling, leading to restoration of GFP expression. A total of twenty-eight hits were identified; elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the most promising. These four hit compounds have EC₅₀ values in the single μM range and three of them (AV-14, -16, -18) represent a chemically related series with an indole structure. We demonstrated that the hit compounds specifically inhibit the STAT2 degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists. At the current time, compounds do not restrict RSV replication in vitro, hence hit optimization is required to improve their potency. Nonetheless, these compounds could be used as chemical tools to determine the unknown mechanism by which NS2 mediates STAT2 degradation and tackle fundamental questions about RSV biology.

No description available.

Les interférons de Type I (IFN) sont des cytokines produites par les cellules en réponse à une infection virale. Les IFNs ont des effets pleïotropiques et parfois paradoxaux, protecteur ou néfaste pour l'immunité Innée ou adaptative. Certains facteurs intrinsèques (type cellulaire) peuvent expliquer une partie ces discordances. Mon travail de thèse s'est intéressé à l'effet du microenvironnement cytokinique sur la réponse IFN. En utilisant des analyses à large échelle, nous avons étudié la réponse IFN dans 4 contextes de polarisation des lymphocytes T auxiliaires (Th). Nous avons identifié 1/ un programme de transcription conservé et 2/ une réponse IFN flexible, modulant spécifiquement les principales fonctions des Th (cytokines, chemokines) en fonction du contexte polarisant. La réponse antivirale apparait aussi flexible avec une moins bonne protection des Th2 et Th17 contre l'infection par HIV-1et HIV-2. Nos résultats suggèrent que l'environnement cytokinique contrôle en partie la réponse IFN et peut ainsi moduler cette dernière dans différents contextes physiopathologiques.

Viral protein U (Vpu) is an accessory protein of HIV‐1 that efficiently targets BST2/Tetherin, a cellular restriction factor that acts as molecular anchor impeding the release of various enveloped viruses from the cell surface. The recently discovered natural receptor of BST2 is ILT7, a molecule exclusively expressed at the surface of the professional type 1 interferon (IFN‐1) producing cells, plasmacytoid dendritic cells (pDCs). The interaction between BST2 and ILT7 has been reported to efficiently induce a repression of IFN­‐1 secretion by pDCs. Here, we investigated the impact of Vpu mediated antagonism of BST2, in regards to this newly described immune function of BST2. Using a system of CD4+ T cell lines infected with wild type or Vpu‐deficient HIV-­1 cultured with peripheral blood mononuclear cells or purified pDCs, we report that the presence of Vpu efficiently reduces IFN-­1 production from sensing pDCs. Furthermore, we observed that this Vpu effect is dependent on the availability of BST2 molecules at the surface of the infected cells, since the Vpu's immunoregulation is abrogated when blocking any potential BST2 trans interaction with anti­‐BST2 antibodies. Similarly, depleting ILT7 from pDCs by means of small interfering RNA treatment equally negates the downregulation of pDC IFN-­1 secretion by Vpu. Finally, the use of recombinant soluble ILT7 competes with pDC‐bound ILT7 for the free BST2 and similarly results in high IFN-­1 production, causing an identical phenotype. Overall, our results demonstrate that Vpu heightens ILT7 activation and subsequent repression of IFN‐1 production by pDCs in response to HIV­‐1 infected CD4+ T cells by promoting it's trans interaction with infected T cell bound BST2, through a yet uncharacterized mechanism. By allowing efficient particle release and restraining pDCs antiviral functions, Vpu exerts a double role on BST2 that seems crucial for the replication and dissemination of HIV‐1. / La protéine accessoire U (Vpu) du VIH‐1 cible efficacement BST2/Tetherin, facteur de restriction restreignant la relâche de divers virus enveloppés à même la surface cellulaire. Le récepteur naturel de BST2 récemment découvert est ILT7, une protéine exclusivement exprimée à la surface des cellules produisant l'essentiel de l'interféron de type 1 (IFN-­1) lors d'infections virales, les cellules dendritiques plasmacytoïdes (pDCs). L'interaction entre BST2 et ILT7 réprime la production d'IFN‐1 des pDCs. Considérant le potentiel immunorégulateur de BST2 récemment décrit, nous avons entrepris d'évaluer cet aspect de l'antagonisme de Vpu sur BST2. À l'aide d'un système de co­‐culture entre une lignée de cellules T CD4+ infectée avec un virus exprimant ou n'exprimant pas Vpu et les cellules mononucléées du sang périphérique ou de pDCs purifiés, nous avons observé que Vpu est responsable d'une atténuation majeure de la sécrétion d'interféron de type 1 (IFN-­1) produite en réponse aux cellules infectées. La présence de molécules de BST2 de surface libres est essentielle à ce processus, puisque le bloc de toute interaction en trans de BST2 par des anticorps polyclonaux α­‐BST2 abroge l'effet de Vpu. Similairement, Vpu ne peut exercer cet effet lorsque ILT7 est déplété dans les pDCs à l'aide de petits ARN interférents. Enfin, l'introduction de protéines recombinantes solubles d'ILT7 dans le système de co-culture semble prévenir l'effet inhibiteur de Vpu, suggérant que Vpu exploite l'interaction de BST2 avec ILT7 pour moduler la sécrétion d'IFN-­1 des pDCs. En conclusion, nos résultats démontrent que Vpu exerce un contrôle sophistiqué de la production d'IFN‐1 par les pDCs en réponse aux lymphocytes T CD4+ infectés par le VIH-­1. Il semble ainsi que l'action de Vpu favorise, par un mécanisme encore méconnu, l'activation d'ILT7 à travers BST2. En effet, cette fonction de Vpu semble tout aussi dépendante de BST2 que de l'ILT7. En favorisant la relâche virale et en menant à l'inhibition de la réponse antivirale des pDCs, la régulation ciblée de BST2 par Vpu est non seulement cruciale à la dissémination du virus, mais aussi à sa réplication.

Diverses études ont souligné le rôle prépondérant du dialogue entre les cellules NK et les cellules dendritiques au cours des réponses immunes. Cependant, les récepteurs impliqués dans ce processus restent incertains. Au cours de ce travail, nous nous sommes attachés à identifier les récepteurs mis en jeu lors de la reconnaissance virale à l’aide de modèles humains et murins. Pour cela, nous avons mimé l’infection virale en utilisant deux ARN bicaténaires synthétiques – poly(AU) et poly(IC) – et montré qu’ils sont tous deux capables d’activer TLR3 mais que seul poly(IC) engage les hélicases RIG-I et MDA5. Les deux ARN induisent l’activation des cellules NK au sein des PBMC humaines, mais seul poly(IC) induit la production d’IFN-gamma. Les DC myéloïdes (mDC) sont requises pour cette activation sans nécessité d’un contact cellulaire entre les cellules NK et les mDC. En outre, les IFN de type I et l’IL-12 secrétés par les DC sont respectivement nécessaires à l’initiation du potentiel lytique et à la production d’IFN-gamma. Poly(IC), au contraire de poly(AU), a une action synergique avec l’IL-12 produite par les mDC pour induire la production d’IFN-gamma en agissant directement sur les cellules NK. Enfin, l’activation conjointe de TLR3 et des hélicases RLR sur les mDC et RIG-I sur les cellules NK, nécessaire à la production d’IFN-gama en réponse à l’ARN bicaténaire, a été confirmée à l’aide de souris déficientes pour TLR3 et Cardif et d’un ligand spécifique de RIG-I. En conclusion, nous rapportons pour la première fois la nécessité pour un composé microbien d’engager deux familles de récepteurs sur deux populations cellulaires distinctes pour induire une réponse innée éfficace. / Crosstalk between NK cells and DC is critical for the response to the microbial mimic poly(IC) but the dsRNA receptors involved in each cell types remained to be defined. We show herein that two dsRNA, poly(AU) and poly(IC), similarly engaged TLR3 while only poly(IC) triggered the RIG-I and MDA-5 helicases. Both dsRNA triggered NK cell activation within PBMC but only poly(IC) induced IFN-gamma. mDC were required for NK cell activation by the two dsRNA, suggesting that they triggered at least TLR3 on mDC. DsRNA induction of cytolytic potential and IFN-gamma production in NK cells did not require contact with mDC but was dependent on the secretion of type I IFN and IL-12, respectively. Poly(IC) but not poly(AU) synergized with mDC-derived IL-12 for high IFN-gamma production by acting directly on NK cells. Finally, the requirement of TLR3 and the RLR on mDC and the involvement of the RIG-I but not TLR3 on NK cells for the production of IFN-gamma induced by dsRNA was confirmed using TLR3 and Cardif deficient mice and RIG-I specific activator. This cooperation was further confirmed using inactivated FLU virus infected-target cells both in human and mouse system demonstrating that NK cells were able to sense viral material by a direct transfer from infected cells likely through lytic immunological synapse without prior infection of NK cells. Thus, we report for the first time the requirement of cotriggering