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1
Daga, Sergio. "CRISPR/Cas9 gene therapy on urine-derived-podocyte-lineage cells and novel biomarker identification: new perspectives in Alport Syndrome (ATS)." Doctoral thesis, Università di Siena, 2019. http://hdl.handle.net/11365/1072659.
Full textAbstract:
Alport syndrome (ATS) is an inherited genetic disorder characterized by glomerular basement membrane (GBM) abnormalities up to end-stage renal disease. Usually, in the most severe forms nephropathy is associated with involvement of eyes and ears because of COL4 chains expression being restricted to kidney, ear and eye. Podocytes, key component of the glomerular structure, are the only cells in the kidney able to produce the COLIVα3-α4-α5 heterotrimer and thus, they are extensively defined as key-players in the pathogenesis of the renal disease.
We have demonstrated how it is possible to isolate podocyte-lineage cells from urines of ATS patients and healthy carriers, providing an easily available cell system closer to podocytes’ physiological conditions.
Our cellular model represents a novel tool and it turned out to be useful not only to characterize the effect of spliceogenic intronic variants but also to identify the presence of cryptic mosaicism confined to the involved tissue and undetectable on peripheral blood samples. This finding dramatically increases the possibility to implement, theoretically with no limits, the molecular genetic test that we can offer to patients.
On the basis of the RNA-Seq data analysis, we have investigated the involvement of IL-32 in ATS.
Being convinced about the fundamental role of IL-32 in ATS, and confirming the real involvement of the IL-32/IL-6/IL-8 pathway in podocytes, we performed the most sensitive ELISA assay to detect IL-32 release into urines of ATS patients. Although no such generous amounts of IL-32 were found in the urines, the values found are reported as sufficient to define a peculiar activation of the IL-32 pathway and more in general inflammatory pathway definitely related to the disease.
At the end, we have explored the innovative gene therapy approach, that we hope will be definitive in the treatment of the disease, directly on the affected cells isolated from patients, exploring the possibility of reverting collagen IV causative mutations in ATS injured podocytes. With this work and with the achieved results we have demonstrated that gene therapy through gene editing approach is not anymore an unexplored frontier, but it can become an increasingly convincing reality. Although preliminary, the achieved results demonstrate how it is possible to obtain a partial recovery of the causative COL4A3 and COL4A5 mutations, unbalancing the heterozygous state towards the wild type condition.
All the results taken together will be fundamental to open new frontiers of management and treatment of the disorder, also in preclinical model, like in dog model through the use of easy-deliverable AAVs system.
2
CARDILLO, MARTINA. "Expression of IL12 and IL23 receptors and cytokines in Chronic Lymphocytic Leukemia and normal B cells." Doctoral thesis, Università degli studi di Genova, 2021. http://hdl.handle.net/11567/1044948.
Full textAbstract:
The mechanisms of clonal expansion of CLL are only partially understood. Several interactions of neoplastic cells with accessory cells and cytokines potentially sustaining neoplastic B cell clone survival and proliferation have been described. Recently, a paracrine/autocrine loop has been reported, involving the upregulation of the IL23R complex and IL23 secretion by CLL cells. This loop drives CLL cell clonal expansion in vitro and in xenografted NSG mice. Furthermore, in situ observations on tissue sections demonstrate that infiltrating IL23 secreting CLL cells interact with macrophages and CD40L expressing T cells. Although inducible in vitro by co-culturing CLL cells with T cells or CD40L expressing cells, the IL23 loop is not observed following stimulation of CLL cells via surface Ig or contact with nurse like cells or bone marrow stromal cells.
In this study, we investigated whether the IL23 loop could be induced following Toll-like receptor 9 (TLR9) engagement which influences leukemic cell survival, activation proliferation albeit in a heterogeneous manner. In addition, we explored the possible existence of an autocrine/paracrine loop mediated by IL12 which shares similarities and surface receptors with IL23 although with a likely opposite outcome in term of the possibility to sustain leukemic cell growth .
IL23R and IL12R complexes (IL23R/IL12Rβ1, IL12β2/IL12Rβ1) expression were evaluated by flow-cytometry following stimulation with CpG oligodeoxynucleotide (ODN) that binds the TLR9 on CLL, showing that CLL cells are able to express the IL23R complex on membrane and, at lower extent, the IL12R complex.
These receptors were assessed also in normal B cells by flow cytometry after 72h of stimulation with CpG and CpG+IL15. In this setting, normal B cells were less capable of IL23R complex expression compared to CLL cells. A further striking difference observed was related to the limited expression of IL12Rß2 receptor chain in stimulated CLL cells compared to normal B cells.
Supernatants of CLL cells and normal B cells were both tested for the production of these cytokines after stimulation. The results showed a low level of IL23p19 secretion for both CLL cells and normal B cells, which is significant after CD40L stimulation (used as positive control), and a higher production of IL12p70 which is more pronounced in normal B cells compared to CLL. In another series of tests, CLL cells were stimulated with CpG for 72h, and subsequently exposed to IL12 or IL23. Exposure to IL12 and IL23 induced the expression of pSTAT1 and pSTAT3. Collectively our data corroborate the notion that IL23R complex act as a pro-survival factor for CLL cells. In contrast, the restricted IL12R complex expression in CLL cells compared to normal B cells indicated that the suppression of the expression of this receptor may favor the survival of the leukemic clones. The possibility of a reciprocal competition of the shared receptor chains is discussed.
3
Pienaar, Sandra Margaret. "Tuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoter." Doctoral thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/9534.
Full textAbstract:
Includes bibliographical references.The aim of this work was to screen the IL12p40 gene promoter for association with TB disease. Initially a subcohort of children (TB cases and healthy controls) from a TB-endemic area was screened for DNA changes by the WAVE method. Thereafter, the entire paediatric cohort and a cohort of healthy adult controls were screened by Amplification Refractory Mutation System PCR. Functional testing was done by reporter assay and immunological phenotype was investigated by measurement of cytokines levels and cytokine receptor expression. WAVE screening identified two heterozygous SNPs, -1523 A/G and -1564 C/T. Statistical analysis showed that -1523 A/G may be protective against TB disease (p=0.02). This possibility was supported by the location of -1523 A/G occurring within a GTATA sequence reported to bind nuclear proteins. Specific ARMS-PCR assays were then designed for screening of additional paediatric subjects and healthy adult controls for these SNPs. Analysis of the larger group, showed that -1564 C/T may contribute to susceptibility to TB disease (p=0.03) Exploring functional relevance, normal and mutant promoter fragments were PCR amplified, using uniquely adapted primers that included restriction sites corresponding to those in the multiple cloning site of an expression vector, facilitating cloning. A truncated promoter and one with essential regions deleted, were created as negative controls. These five promoter fragments were cloned into the expression vector and functional differences tested by reporter. No significant functional differences between variant and normal promoter fragments were observed. A predictive immune phenotype was investigated by measurement of IFNγ, TNFα and IL12p70 cytokine levels and IL12βR1 receptor expression. While distinct patterns of cytokine responses were seen, these did not predict genotype. These results show that the IL12p40 gene promoter is highly conserved and sequence variants may be just one of many factors contributing to TB susceptibility.
4
Perri, Graziela. "Presença de IL33 em amostras de carcinoma espinocelular." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/25/25149/tde-30032017-213204/.
Full textAbstract:
O carcinoma espinocelular (CEC) é a segunda forma de neoplasia cutânea mais prevalente. Os mecanismos exatos envolvidos na progressão desse tipo de tumor ainda não estão elucidados. Estudos recentes têm mostrado que a citocina IL33 é uma citocina reguladora da resposta imune adaptativa, principalmente como potente indutor do perfil Th2. Juntamente com seu receptor ST2, apresenta-se com os níveis elevados em alguns tipos de câncer, corroborando para a evidência de que essa citocina contribui para a carcinogênese. Baseado nessas informações, testamos a hipótese de que a presença de IL33 em carcinoma espinocelular, poderia estar relacionada a um melhor prognóstico. Neste estudo foram utilizadas amostras de carcinoma espinocelular, em diferentes gradações de malignidade tumoral (Grau I, Grau II e Grau III). Os resultados mostraram um infiltrado inflamatório mais intenso em tumores com Grau I e II. Imunorreatividade para IL33 foi observada em tumores de Grau I e II tanto por células epiteliais como por células do infiltrado inflamatório. A análise por microscopia confocal evidenciou que um grande número de células TCD4+ e TCD8+ que expressavam IL33 foi observado em tumores de Grau II. Esses resultados indicam a presença de um intenso infiltrado inflamatório e expressão de IL33 em amostras de carcinoma espinocelular com níveis menores de malignidade tumoral.Squamous cell carcinoma (SCC) is the second most common form of cutaneous neoplasm. The exact mechanisms involved in the progression of this type of tumor have not yet been elucidated. Recent studies have shown that the cytokine IL33 is a cytokine regulating the adaptive immune response, mainly as a potent inducer of Th2 profile. Together with its ST2 receptor, its presents with elevated levels in some types of cancer, corroborating to evidence that this cytokine contributes to carcinogenesis. Based on this information, we tested the hypothesis that the presence of IL33 in squamous cell carcinoma could be related to a better prognosis. In this study, squamous cell carcinoma samples were used in three different gradations of tumor malignancy (Grade I, Grade II and Grade III). The results showed that a more intense inflammatory infiltrate in Grade I and II tumors. Immunoreactivity for IL33 was observed in Grade I and Grade II tumor, by epithelial cells and by inflammatory infiltrate cells. The analysis by confocal microscopy evidenced that a great number of TCD8+ and TCD4+ cells expressing IL33 was observed in grade II tumors. These results indicate the presence of an intense inflammatory infiltrate and expression of IL33 in samples of squamous cell carcinoma with lower levels of tumor malignancy.
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Ruiz, Castilla Mireia. "Paper de l’eix IL33/ST2 en el pacient cremat." Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/665727.
Full textAbstract:
La lesió per cremada s’ha associat a l’augment de la concentració de molts mediadors inflamatoris. Aquests mediadors són importants en la fisiopatologia de la cremada, contribuint a la disfunció orgànica i l’aparició de complicacions sèptiques. També són útils en l’establiment del pronòstic i són importants en la fisiopatologia de situacions concretes com la cicatrització o la lesió per inhalació de fums. En conseqüència, alguns d’aquests biomarcadors també podrien ser considerats com a possibles dianes terapèutiques. Igualment, com que els tractaments també poden afectar els processos biològics, els biomarcadors poden ser útils per guiar l’ús de determinats tractaments i podrien ajudar a explicar perquè alguns tractaments no són útils a l’hora de millorar el pronòstic de determinats pacients. Per tant, la investigació en biomarcadors és una característica principal de la medicina translacional d’aquesta àrea de coneixement.
La present tesi té l’objectiu de valorar la utilitat en la determinació del pronòstic dels pacients cremats dels biomarcadors implicats en l’eix IL33/ST2. De fet, es tracta del primer article que analitza la significació pronòstica d’aquests biomarcadors en pacients cremats i els resultats obtinguts demostren la relació existent entre la concentració de la fracció soluble de la proteïna supressió de la tumorigenicitat 2 (sST2) i la mortalitat d’aquests pacients. A més a més, nivells elevats de sST2 també es van associar a una major incidència de complicacions infeccioses i de disfunció orgànica, suggerint que podria tenir un paper significatiu en la gènesi de la disfunció orgànica associada a la cremada. Per tots aquests motius, la mesura de la concentració de sST2 podria, en un futur, ajudar en el procés de presa de decisions sobre el tractament indicat en cada pacient ja que ens permetria saber quins són els pacients amb més alt risc de patir una mala evolució i que, per tant, es podrien beneficiar d’un tractament més agressiu.Several inflammatory mediators have been shown to be increased after burn injury. They may be important in burn pathophysiology, contributing to organ dysfunction and sepsis apparition, and they may also predict outcomes. Moreover, they have been involved in pathophysiology of some special processes, such as inhalation injury or wound healing. Consequently, some biomarkers have been described as potential therapeutic targets. Importantly, as therapeutic interventions may also affect biological processes, biomarkers may be a useful tool to guide some treatments and may also explain why some treatments succeed or fail in improving outcomes. Therefore, investigation into biomarkers in severe burn patients is a key feature of translational medicine in this area of knowledge. Our aim was to analyze whether plasma levels of biomarkers involved in the IL33/ST2 axis might help to predict mortality in burn patients. This is the first study to show the prognostic significance of plasma levels of sST2 after burn injury. Indeed, higher plasma concentrations of sST2 were consistently associated with a higher risk of death, even after adjusting for different potential confounding. Moreover, higher levels of sST2 were also observed in burn patients who developed any infectious complication during their stay in the Burns Unit as well as in patients who presented MODS. In conclusion, the results of this study suggest that plasma sST2 levels predict mortality in burn patients and may be useful to help or guide physicians in the bedside decision-making process during patient management.
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Verma, Akash. "Unraveling the IL4-IL33 Nexus in Histoplasma Capsulatum Infection." University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1406898828.
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Ferrasi, Adriana Camargo [UNESP]. "Transcript finishing initiative: contribuição do laboratório IL2." Universidade Estadual Paulista (UNESP), 2003. http://hdl.handle.net/11449/87745.
Full textAbstract:
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ferrasi_ac_me_rcla.pdf: 2629180 bytes, checksum: c178eeb179f2c77ab45bfcddeb648f51 (MD5)O principal objetivo na análise de um genoma é a identificação gênica. Várias ferramentas computacionais estão disponíveis para este propósito e são baseadas em similaridade (BLAST e BLAT) ou em predição de genes (Genscan e Fgenes). Entretanto, estes programas estão se mostrando ineficientes para detectar e caracterizar todos os genes presentes no genoma humano. A importância das informações de cDNAs tem sido reconhecida desde o início do Projeto Genoma Humano, entretanto, o seqüenciamento em larga escala de cDNAs completos ainda requer técnicas avançadas tais como a produção de bibliotecas de cDNAs enriquecidas por transcritos grandes e raros. O seqüenciamento parcial de etiquetas de seqüências expressas (ESTs) foi desenvolvido como uma técnica alternativa para gerar, em larga escala, vários tipos de cDNAs. Atualmente, a maioria das informações de cDNAs no GenBank são representadas por ESTs convencionais 3þ e 5þ e ORESTES (provenientes das porções centrais dos transcritos). Baseados nos bancos de dados gerados pelo alinhamento de todas essas seqüências com as seqüências genômicas humanas disponíveis foi proposta a estratégia transcript finishing para a caracterização e validação de novos genes humanos, como parte do consórcio entre FAPESP e Instituto Ludwig de Pesquisa sobre o Câncer. O projeto Transcript Finishing Initiative está sendo realizado por uma rede de 31 diferentes grupos de pesquisa do Estado de São Paulo. Foram selecionados pela coordenação do projeto, 602 transcritos e destes 300 (50%) foram validados. Destes transcritos, 20 foram atribuídos ao laboratório validador IL2, e destes, 11 (55%) foram validados. Utilizando ferramentas de bioinformática, o laboratório IL2 realizou uma anotação preliminar dos consensos de seus transcritos validados (disponibilizados pela coordenação do projeto)... .
A fundamental task in analyzing genome is gene identification. This is relatively straightforward for compact genome but much more challenging for complex genomes. Some computational tools are available for this purpose, but they are bases on similarity (BLAST) or prediction analysis (Genscan and Fgenes). However, these programs are inefficient to detect and characterize all genes present in the genome. The importance of cDNA information has been recognized since the beginning of the Human Genome Project, however cost-effective and hightroughput sequencing of full-length cDNA still requires technical advances such as the production of cDNA libraries enriched for large and rare transcripts. Partial sequencing of expressed sequences (EST) has been developed as an alternative approach for the generation, in large-scale, of several kinds of cDNAs. Currently, the vast majority of cDNA data in the GenBank is represented both by conventional 5þand 3þexpressed sequence tags (ESTs) and by ORESTES (open reading frame ESTs), which is derived from central portions of the transcripts. Based on a database generated through alignment of all of these sequences to the available human genomic sequences, have been proposed the transcript finishing strategy for characterization and validation of new human genes, as part of the FAPESP-LICR Transcript Finishing Initiative. The strategy utilizes the ORESTES scaffold EST sequence to build primers for reverse transcription (RT) - PCR reactions in order to bridge gaps, thereby confirming the membership of ESTs to a common transcript and providing information on the intervening sequence (validation strategy). The FAPESP-LICR Transcript Finishing Initiative is being pursed by a network of 31 different research groups from the State of São Paulo (The Transcript Finishing Consortium) coordinated by 2 different laboratories... (Complete abstract click electronic address below).
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Ferrasi, Adriana Camargo. ""Transcript finishing initiative" : contribuição do laboratório IL2 /." Rio Claro : [s.n.], 2003. http://hdl.handle.net/11449/87745.
Full textAbstract:
Orientador: Maria Inês de Moura Campos PardiniBanca: Maurício Bacci Junior
Banca: Magaly Machado Sales
Resumo: O principal objetivo na análise de um genoma é a identificação gênica. Várias ferramentas computacionais estão disponíveis para este propósito e são baseadas em similaridade (BLAST e BLAT) ou em predição de genes (Genscan e Fgenes). Entretanto, estes programas estão se mostrando ineficientes para detectar e caracterizar todos os genes presentes no genoma humano. A importância das informações de cDNAs tem sido reconhecida desde o início do Projeto Genoma Humano, entretanto, o seqüenciamento em larga escala de cDNAs completos ainda requer técnicas avançadas tais como a produção de bibliotecas de cDNAs enriquecidas por transcritos grandes e raros. O seqüenciamento parcial de etiquetas de seqüências expressas (ESTs) foi desenvolvido como uma técnica alternativa para gerar, em larga escala, vários tipos de cDNAs. Atualmente, a maioria das informações de cDNAs no GenBank são representadas por ESTs convencionais 3þ e 5þ e ORESTES (provenientes das porções centrais dos transcritos). Baseados nos bancos de dados gerados pelo alinhamento de todas essas seqüências com as seqüências genômicas humanas disponíveis foi proposta a estratégia "transcript finishing" para a caracterização e validação de novos genes humanos, como parte do consórcio entre FAPESP e Instituto Ludwig de Pesquisa sobre o Câncer. O projeto "Transcript Finishing Initiative" está sendo realizado por uma rede de 31 diferentes grupos de pesquisa do Estado de São Paulo. Foram selecionados pela coordenação do projeto, 602 transcritos e destes 300 (50%) foram validados. Destes transcritos, 20 foram atribuídos ao laboratório validador IL2, e destes, 11 (55%) foram validados. Utilizando ferramentas de bioinformática, o laboratório IL2 realizou uma anotação preliminar dos consensos de seus transcritos validados (disponibilizados pela coordenação do projeto)... (Resumo completo, clicar acesso eletrônico abaixo).
Abstract: A fundamental task in analyzing genome is gene identification. This is relatively straightforward for compact genome but much more challenging for complex genomes. Some computational tools are available for this purpose, but they are bases on similarity (BLAST) or prediction analysis (Genscan and Fgenes). However, these programs are inefficient to detect and characterize all genes present in the genome. The importance of cDNA information has been recognized since the beginning of the Human Genome Project, however cost-effective and hightroughput sequencing of full-length cDNA still requires technical advances such as the production of cDNA libraries enriched for large and rare transcripts. Partial sequencing of expressed sequences (EST) has been developed as an alternative approach for the generation, in large-scale, of several kinds of cDNAs. Currently, the vast majority of cDNA data in the GenBank is represented both by conventional 5þand 3þexpressed sequence tags (ESTs) and by ORESTES (open reading frame ESTs), which is derived from central portions of the transcripts. Based on a database generated through alignment of all of these sequences to the available human genomic sequences, have been proposed the transcript finishing strategy for characterization and validation of new human genes, as part of the FAPESP-LICR Transcript Finishing Initiative. The strategy utilizes the ORESTES scaffold EST sequence to build primers for reverse transcription (RT) - PCR reactions in order to bridge gaps, thereby confirming the membership of ESTs to a common transcript and providing information on the intervening sequence (validation strategy). The FAPESP-LICR Transcript Finishing Initiative is being pursed by a network of 31 different research groups from the State of São Paulo (The Transcript Finishing Consortium) coordinated by 2 different laboratories... (Complete abstract click electronic address below).
Mestre
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Nicol, Louise Maureen Marie. "Investigating differential T cell polarization in the two pathological forms of sheep paratuberculosis." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/22855.
Full textAbstract:
Paratuberculosis is a chronic enteropathy of ruminants that presents as two distinct disease forms in sheep; paucibacillary (or tuberculoid) and multibacillary (or lepromatous) disease. The immunopathology of paucibacillary and multibacillary sheep paratuberculosis has been linked to inflammatory Th1/Th17 cell and Th2/macrophage responses respectively. IL23 and IL25 are key to the development of these responses by interaction with their complex receptors, IL23R/IL12RB1 and IL17RA/IL17RB. Furthermore, the polarization of T cells and the development of appropriate immune responses is controlled by the master regulator transcription factor; T-bet, GATA3, RORγt and RORα. In humans, variations in the structure, sequence and/or expression of the genes encoding these proteins have been implicated in the different pathological forms of tuberculosis and leprosy, and gastrointestinal inflammatory disorders such as Crohn’s disease. In the current study, sequencing has identified multiple transcript variants of sheep IL23R, IL12RB1 and IL17RB and a single IL17RA transcript. RT-qPCR assays were developed for the cytokine receptor variants identified in this study and known transcript variants of the transcription factor genes. Expression levels were compared in the ileo cecal lymph node of paucibacillary or multibacillary paratuberculosis diseased sheep. Of the cytokine receptors; the IL12RB1v3 variant, which lacks the receptor activation motif, was differentially expressed and was significantly increased in multibacillary disease; this may contribute to high Th2 responses. Full length IL17RB was differentially expressed and was significantly increased in multibacillary pathology, which may also contribute to Th2 polarization. IL17RA was significantly increased in paucibacillary disease. The contrast between the IL17RA and IL17RB results may indicate that, in addition to Th1 cells, Th17 T cells are also involved in paucibacillary pathology. Of the transcription factor transcripts; full length TBX21 (T-bet) was differentially expressed and was significantly increased in paucibacillary disease; this may explain increased Th1 responses in these sheep. Full length GATA3 was significantly increased in paucibacillary compared to multibacillary sheep, suggesting a loss of Th2 responses in late-stage multibacillary pathology. RORAv1 variant was differentially expressed and was significantly increased in paucibacillary pathology, indicating a role of Th17 T cells in paucibacillary pathology.
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Geremia, Alessandra. "The role of the IL23/IL17 pathway in inflammatory bowel disease." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:f39c2ab5-098e-45d8-a800-e4c4bc7ae85f.
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The aetiology of IBD is unknown, but available evidence suggests that an aberrant immune response towards the commensal microbial flora is responsible for intestinal inflammation in genetically susceptible individuals. Studies from animal models of intestinal inflammation have greatly advanced our understanding of the immunological basis of IBD. However, translation of results from animal research into human studies is essential in order to improve treatment options and patient quality of life. In this thesis we present the successful introduction of translational studies on human tissue in our laboratory. In particular, we evaluated the role of the IL23/IL17 pathway in the human immune response and its role in IBD. IL23-driven inflammation has been primarily linked to its activity on Th-17 cells; however, work from our laboratory has identified a novel population of IL23-responsive ILC, which are responsible for innate colitis in mice. Here we have analyzed the role of IL23-responsive innate cells in IBD. Our results show increased expression of Th-17 signature genes amongst intestinal CD3- cells in patients with IBD. Furthermore, we observed a marked and selective increase in IL17 producing CD56- ILC in the inflamed intestine of patients with CD. ILC may contribute to intestinal inflammation through secretion of cytokines, such as IL17A and IL17F, and recruitment of other inflammatory cells, representing a novel tissue-specific target for the treatment of IBD. In addition, we present here our preliminary data on the characterization of human intestinal and systemic DC populations. In particular, we aimed to evaluate if in the context of the intestinal microenvironment DC develop specific regulatory features, as observed in murine CD103+ DC. We show that human intestinal DC populations exhibit specific regulatory properties, such as expression of genes associated with TGF-β and RA activity. Furthermore, CD103+ DC are present in the human gut and are characterized by tolerogenic markers. Remarkably, patients with IBD have reduced frequencies of intestinal CD103+ DC, which display a more pro-inflammatory phenotype. Alteration in DC subset composition and functional activity may result in a distort balance between immune effector and regulatory responses, promoting the development of intestinal inflammation.
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Queiroz, Gerson de almeida. "Estudo da influência de polimorfismos em il33 e il1rl1 na asma." Universidade Federal da Bahia, 2016. http://repositorio.ufba.br/ri/handle/ri/21383.
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Asma e atopia são condições determinadas por fatores ambientais e genéticos Vários estudos de associação do genoma têm sido realizados para tentar entender os componentes genéticos de tais condições. Os genes IL33 e IL1RL1 são os mais replicados em estudos do tipo GWAS em todo o mundo. A citocina IL-33 e o seu receptor ligado à membrana (ST2L) ou sua forma solúvel (sST2), em conjunto são potentes moduladores de inflamação do tipo Th2. Quando ligada ao ST2L, IL-33 produzida por múltiplas células da resposta inata e adaptativa, induz citocinas pró-inflamatórias, tais como IL-4, IL-5 e IL-13, aumentando a resposta e inflamação Th2, principal característica da asma e alergias. Por outro lado, quando a IL-33 se liga ao sST2, neutraliza o seu efeito, impedindo sua ligação ao ST2L. Vários polimorfismos nestes genes têm sido associados com a asma e atopia. Assim, os fatores genéticos que afetam IL33 e IL1RL1 podem influenciar a susceptibilidade para asma e atopia. Neste contexto, este estudo teve como objetivo avaliar a influência de polimorfismos em IL33 e IL1RL1 na asma e atopia em uma população Latina. Estratégias diferentes foram usadas para, um estudo de coorte de asma leve e um estudo caso-controle de asma grave. O alelo A para rs1041973 em IL1RL1 na coorte SCAALA foi positivamente associado com IL-5 produção (OR 1,36, IC 95% 1,09-1,84, P=0,044) IgE específica (OR 1,40, IC 95% 1,07-1,84, P=0,013) e SPT (OR 1,48, IC 95% 1,08-2,03, P=0,014), ambos contra o ácaro B. tropicalis. Além disso, indivíduos atópicos com o genótipo AA de rs1041973 mostraram uma diminuição da produção de sST2 comparado com indivíduos com os genótipos AC e CC (P <0,05). O alelo G do SNP IL33 rs12551256 foi negativamente associado com asma (OR 0,71, IC 95% 0,53-0,94, P=0,017). Em relação ao estudo caso controle do ProAR, o alelo A do rs1420101 em IL1RL1, foi positivamente associado com asma atópica (OR 1,29, IC 95% 1,05-1,66, P=0,046) e negativamente com FEV1 (BETA -2,37, IC 95% -4,67; -0,07, P=0,043). Além disso, este mesmo alelo mostrou uma diferença estatisticamente significate com uma menor produção de sST2 plasmático em indivíduos controle (P <0,001). O alelo C do rs2381416 foi positivamente associado com SPT para A. flavus. (OR 7,2, IC 95% 1,05-3,37, P=0,033), epitélio de cão (OR 1,52, IC 95% 1,19-3,47, P=0,009) e gato (OR 1,52, IC 95% 1,04-2,63, P=0,048). Estes dados sugerem que os SNPs nos genes IL33 e IL1RL1 podem ter um impacto sobre o desenvolvimento de asma e alergia na população brasileira. No entanto, mais estudos devem ser realizados para elucidar o impacto funcional de tais polimorfismos aqui descritos no desenvolvimento de asma e atopia.
QUEIROZ, Gerson de Almeida. STUDY OF INFLUENCE OF IL33 AND IL1RL1 POLYMORPHISMS IN SEVERE ASTHMA. 93f. 2016. Dissertação (Mestrado) - Instituto de Ciências da Saúde, Universidade Federal da Bahia. Asthma and atopy are conditions determined by environmental and genetic factors. Several genome-wide association studies have been conducted to try to understand the genetic components of such conditions. The IL33 and IL1RL1 are the most replicated genes in GWAS studies worldwide. The cytokine IL-33 and its receptor, membrane bound (ST2L) or its soluble form (sST2) together are potent Th2-type inflammation modulators. When bound to ST2L, IL-33 produced by multiple cells of the innate and adaptive response, induce proinflammatory cytokines such as IL-4, IL-5 and IL-13, increasing the response and Th2 inflammation main feature of asthma and allergies. On the other hand, when IL-33 binds to sST2, neutralizes their effect by preventing its binding to ST2L. Several polymorphisms in these genes have been associated with asthma and atopy. Thus, genetic factors that affect IL33 and IL1RL1 may influence susceptibility to asthma and atopy. In this context, this study aimed to evaluate the influence of polymorphisms in IL33 and IL1RL1 in asthma and atopy in a Latino population. To verify that he have used to different strategies, a cohort study for mild asthma and a case-control study for severe asthma. The A allele for rs1041973 in IL1RL1 in SCAALA cohort was positively associated with IL-5 production (1.36 OR, 95% CI 1.09-1.84, p=0.044) specific IgE (OR 1.40, 95% CI 1.07-1.84, p=0.013) and SPT (OR 1.48, 1.08-2.03 95% CI, p=0.014), both against B. tropicalis mite. Furthermore, atopic individuals with the AA genotype of rs1041973 showed a decreased production of sST2 as compared to individuals with the AC and CC genotypes (P<0.05). The G allele of IL33 SNP was negatively associated with asthma (OR 0.71, 95% CI 0.53-0.94, p=0.017). Regarding the ProAR case control study the A allele of rs1420101 in IL1RL1 was positively associated with atopic asthma (OR 1.29, 95% CI 1.05-1.66, P=0.046) and negatively associated with FEV1 (BETA -2.37, 95% CI -4.67 ; -0.07, P=0.043). In addition, this same allele showed a statistically significant difference with a lower production of plasma sST2 in control subjects (P<0.001). The C allele of rs2381416 was positively associated with SPT to A. flavus. (OR 7.2, 95% CI 1.05 to 3.37, P = 0.033), dog (OR 1:52, 1:19 to 3:47 95%, P = 0.009) and cat epithelium (OR 1:52, 95% CI 1.04-2.63, P = 0.048). These data suggest that SNPs in IL33 and IL1RL1 genes may have an impact on the development of asthma and allergy in Brazilian population. However, further studies should be conducted to further elucidate the functional impact of such polymorphisms described herein in the development of asthma and atopy.
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Vernisse, Charlotte. "Cellules Club et susceptibilité respiratoire environnementale." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTT007.
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La BPCO (BronchoPneumopathie Chronique Obstructive) est une maladie chronique des voies aériennes où prédominent modifications structurelles et inflammation. Le but de ce travail a été d’étudier différents axes caractéristiques de cette pathologie comme l’hyperplasie des cellules à mucus, le déficit en cellules Club et la dérégulation des alarmines pour trouver de nouvelles cibles thérapeutiques.Tout d’abord, nous avons réalisé la carte d’identité des cellules Club issues de cultures en interface air-liquide (ALI) de cellules épithéliales bronchiques humaines par deux méthodes (Single cell-RNAseq et puces à ADN). Les résultats obtenus en Single cell-RNA seq montrent que la totalité des cellules épithéliales expriment l’ARNm CCSP. Pour pallier à ce problème, nous avons triées les cellules selon l’expression de leur protéine spécifique SCGB1A1. Les puces à ADN ont identifié trois populations exprimant la protéine CCSP (fluorescence et granulosité différentes). Ces différentes populations ont une expression d’ARNm de type ciliée ou de type classique « Club ». Ceci permet de montrer la plasticité de ces cellules et leur potentiel pour rétablir un épithélium différencié. Ensuite, nous avons montré que les voies de signalisation BMP et Notch sont déterminantes pour le devenir des cellules épithéliales des voies aériennes. Par conséquent, il est possible de maîtriser la différenciation des cellules provenant de cultures ALI NHBE et HBEC en cellules ciliées, caliciformes, Club ou basales. Un inhibiteur de Notch, le DapT, permettait de rétablir le déséquilibre caractéristique de la BPCO en favorisant le phénotype cilié, mis en évidence aussi bien en immunofluorescence qu’en single cell RNAseq. A partir des données obtenues, il est possible d’envisager de superviser le phénotype des cellules épithéliales des voies aériennes. Pour finir, nous avons étudié différents profils de sécrétions d’alarmines dans les cultures ALI de cellules épithéliales bronchiques humaines à l’état basal. TSLP et IL33 apparaissaient augmentées dans la BPCO, tandis que l’IL25 était diminuée comparé aux sujets sains et aux asthmatiques. Ces patterns d’expression étaient associés de manière hétérogène aux marqueurs cliniques de l’inflammation de type 2. Pour conclure, il n’y a donc pas une mais des cellules Club, aux morphologies, profils transcriptomiques et fonctions différentes, engagées vers des voies de différenciation distinctes. Le relatif maintien d’un profil cytokinique de type T2 d’un épithélium cultivé en ALI en relation avec les données cliniques valide ce modèleCOPD (Chronic Obstructive Pulmonary Disease) is characterized primarily by parenchymal structural changes and inflammation. The aim of this work was to study different characteristics of this pathology such as mucus cell hyperplasia, Club cell deficits and alarmin deregulation in order to find new therapeutic targets. First, we used two transcriptomic methods (Single cell-RNAseq and DNA chips) on air-liquid interface (ALI) cultures of human bronchial epithelial cells to produce a Club cell “identity card”. Single cell-RNA seq results demonstrated that all epithelial cells express mRNA for Club cell secretory protein (CCSP), the biomarker classically used for identifying Club cells. To overcome this ubiquity problem, we sorted the cells according their relative level of SCGB1A1 (the gene encoding CCSP) protein expression. DNA chips identified three CCSP-expressing cell populations differing by fluorescence level and/or scatter. These different populations expressed not only classic "Club" mRNA transcripts, but also markers associated with ciliated cells, highlighting their plasticity and potential to restore a differentiated epithelium. We then demonstrated that the BMP and Notch signaling pathways determine the fate of airway epithelial cells, consequently enabling differentiation of cells from ALI NHBE and HBEC cultures into ciliated, goblet, Club or basal cells. A Notch inhibitor, DapT, restored the characteristic imbalance of COPD by promoting the ciliated phenotype, (demonstrated both by immunofluorescence and via single cell RNAseq). It is therefore now possible to control the phenotype of the airway epithelial cells in culture. Finally, we studied different alarmin secretion profiles in ALI cultures of HBECs at basal state. TSLP and IL33 appeared to be increased in COPD, while IL25 was decreased compared to healthy subjects and asthmatics. These expression patterns were heterogeneously associated with clinical markers of type 2 inflammation.In conclusion, there is not a single Club cell, but multiple types of Club cells with different morphologies, transcriptomic profiles and functions that are committed to different differentiation pathways. The relative maintenance of a T2 cytokine profile of the epithelial cells cultivated in ALI in accordance with clinical data strongly supports the validity of the model
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Balmas, Elisa. "Group 2 innate lymphoid cells and reproduction." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/271310.
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Regulation of the immune system and of uterine tissue homeostasis, growth, and remodelling are deeply intertwined during pregnancy and are essential for successful reproduction. Recent findings showed that tissue-resident innate lymphoid cells (ILCs) are crucial regulators of both physiology and pathology of the tissues they populate. Uterine natural killer (uNK) cells are a subtype of ILCs known to regulate trophoblast invasion, uterine vascular adaptation to pregnancy, and foetal growth. We recently described additional types of ILCs in the uterus of women and mice. However, the role of these ILCs during reproduction is unknown. Among them, group 2 ILCs (ILC2s) have been previously characterised in other tissues, in which they modulate immune cells and tissue homeostasis by producing type-2 cytokines and growth factors (i.e. IL-4, IL-5, IL-13, and Amphiregulin). Based on these premises, I hypothesized that uterine ILC2s (uILC2s) regulate uterine immune homeostasis and thus contribute to successful reproduction. To test this, I first characterised the uILC subtypes present in humans and mice at various stages of the reproductive cycle. Secondly, I addressed the functional role of uILC2s during pregnancy by taking advantage of a uILC2 knockout mouse model. My results show that uterine ILC2s represent < 1% and < 0.1% of murine and human uterine leukocytes, respectively. However, as they can quickly produce large amounts of cytokines, uILCs are capable of potently affect both other immune cells and the surrounding tissue. Indeed, I found that compared to other tissue-resident ILC2s, uILC2s produce high levels of IL-5 and Areg even in the absence of any stimulation. On the contrary, non-uterine ILC2s mainly produce IL-13, which is lowly expressed by uILC2s. To further characterize the tissuespecific properties of uILC2s, I then performed RNAseq on uILC2s isolated from virgin, midgestation, and term murine uterus, and I compared their transcriptomes with those of ILC2s from lung, intestine, and bone marrow. Interestingly, uILC2s specifically express granzymes and genes typical of regulatory T cells. Therefore, uILC2s have tissue-specific properties and are modulated during pregnancy. Furthermore, the ability of uILC2s to produce IL-5 and Areg suggests that they may be crucial in the regulation of uterine type-2 immunity. I then studied the phenotype of $Rora^{flox/flox}Il7ra^{cre/wt}$(ILC2KO) mouse models, as well as that of mice lacking the ILC2 activating cytokine IL-33 ($IL33^{cit/cit}$; IL33KO). I examined the immune microenvironment in both the myometrium and decidua in ILC2KO mice and found alterations in type-2 cytokines and myeloid cell homeostasis. In particular, in absence of ILC2s, IL-4 and IL-5 are dramatically reduced, IL-13 is absent, and decidual inflammatory cytokines IL1β and IL-6 are increased. Furthermore, uterine dendritic cells (uDC), uterine macrophages (uMac), and uterine neutrophils (uN) increase, while uterine eosinophils (uEo) are virtually absent. These results show that uILC2s regulate uterine type-2 immunity, suggesting that uILC2s could be important during pregnancy. Accordingly, I found that lack of uILC2s leads to insufficient spiral artery remodelling and restricted foetal growth. Type-2 cytokines and in particular IL-4 regulates alternative activation of Macrophages (Mac) and Dendritic Cells (DCs), which promote the development of an anti-inflammatory environment and facilitate tissue remodelling. I hypothesised that similar mechanisms occur in the uterus and that uILC2s have a central role in the polarisation of the immune response. To explore this, I studied in more detail the characteristics of uEo, uMac, and uDCs dissected from wild type and ILC2KO mice. I found a reduction in genes associated with alternative activation in uMac and uDCs in the uterus of pregnant ILC2KO mice. Additionally, I showed that uEo are the main producers of the IL-4. This demonstrates that uILC2s promote alternative activation of myeloid cell population by modulating the uterine immune microenvironment. I then assessed the role of uILC2s-dependent type-2 immunity in inflammatory pathology following a type-1 response to bacterial infection. When challenged with LPS, pregnant ILC2KO mice showed more pronounced foetal demise. Therefore, uILC2s regulate uterine type-2 immune homeostasis and this prevents inflammatory pathology. Collectively, my work advances our knowledge of the innate immune mechanisms that control physiological and pathological events during pregnancy. These findings have implications to the field of immunology of pregnancy and may lead to clinical progress in diagnosis and prevention of infection-induced abortion in human pregnancies.
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Piatek, Stefan. "Functional genomics of the major asthma gene IL33 in airway epithelial cells." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/55280.
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Asthma is a common disease of the large airways and the airway epithelium has a sentinel role in the initiation of the disease. Interleukin-33 is located in the nucleus of airway epithelium and is increased in asthmatic airway epithelium. Furthermore the gene has been reproducibly associated with asthma by genome wide association studies. Originally identified as a nuclear factor, interleukin-33 was found to act as a cytokine via the receptor ST2. Since this point the majority of research on interleukin-33 has focused on its role as a cytokine. Studies of its nuclear role have investigated the expression of a handful of inflammatory genes. This thesis examines the role of nuclear interleukin-33 in the airway epithelium using a functional genomics approach. The alveolar A549 cell line and normal primary bronchial epithelial cells were found to express full-length interleukin-33 mRNA. Both were taken forward for interleukin-33 knockdown studies using enzyme-linked immunosorbent assays to measure relevant cytokines in supernatants. Upon interleukin-33 knockdown, interleukin-1ß-induced interleukin-6 and interleukin-8 secretion was reduced in both A549 and primary cells. Microarray analyses of gene expression showed interleukin-33 affected genes involved in differentiation, cell-substrate adhesion and extracellular matrix organisation. In contrast, ST2 knockdown resulted in increases in cytokine secretion upon interleukin-1ß stimulation with global gene expression analyses showing little overlap with interleukin-33-effected genes. Taken together, this suggests that the gene expression changes found upon interleukin-33 knockdown were due to a nuclear role. Finally, chromatin-immunoprecipitation sequencing was used to determine genome wide the DNA-binding sites of interleukin-33. Transcription factor binding motifs and preferential binding near promoters and exons were found but further replicates are required for confidence in these results. This work shows that interleukin-33 has an important nuclear role that appears to regulate gene expression in a manner that could mediate the changes seen in the asthmatic lung.
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Ward, Jacqueline Suzanne. "The expression, regulation and function of endothelial IL2 receptors /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487864986611013.
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Bains, Navdeep Kaur. "Expression of 9-cis-epoxycarotenoid dioxygenase and ILL2 : enzymes of plant hormone biosynthesis." Thesis, Queen Mary, University of London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429148.
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Petit, Maxime. "Residency and trafficking of ILC2 in steady steate and th2 induced inflammatory conditions." Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7095.
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Les ILC2s sont retrouvées au niveau des muqueuses comme les poumons et l’intestin, ainsi que dans divers ganglions et organes liés au métabolisme comme les tissus adipeux (ATs). Elles jouent un rôle important dans l’induction des réponses immunitaires de type Th2 comme équivalents innées dans lymphocytes Th2. Elles sont activées par des alarmines (IL-25 et IL-33) et des activateurs environnementaux (allergènes, métabolites et neuromédiateurs). Les ILC2s sécrètent des cytokines de type Th2 permettant de recruter et d’activer des cellules myéloïdes, d’augmenter la production de mucus et la contraction musculaire, ainsi que d’initier la réparation et le renouvellement des tissus. Cependant, une activation non contrôlée des ILC2s participe au développement de maladies chroniques. Les ILCs sont généralement considérées comme des cellules résidentes. Cependant, plusieurs études ont suggéré que la migration pourrait être un processus important pour la maturation des capacités effectrices. La circulation des ILCs reste peu documentée, et aucun mécanisme n’est pour l’instant capable d’expliquer le renouvellement des ILC2s pour agir dans de nombreux tissus suite à une stimulation. Nous avons montré que des quantités significatives d’ILC2s matures et immatures peuvent être collectées dans la lymphe du canal thoracique de souris canulées durant plusieurs heures. Les ILC2s circulantes forment 3 groupes distincts avec des expressions de molécules d’adhésion et récepteurs de migration spécifiques. Nos expériences de transferts cellulaires montrent que ces groupes spécifiques de molécules exprimées sont liés à des tropismes particuliers pour l’intestin, les poumons ou les ATs. Pour analyser le comportement des ILC2s dans un contexte de réponse de type Th2, nous avons injecter les cytokines IL-25 et IL-33 et étudié la lymphe de ces souris. La stimulation à l’IL-33 augmente le nombre de cellules ILC2s circulants dans la lymphe. Les différents groupes d’ILC2s montrent des réponses différentes à l’IL-33. Ainsi, les ILC2s migrants vers l’intestin sont majoritairement prolifératives tandis que le groupe migrant vers les poumons et les ATs secrètent de l’IL-5, de l’IL-13 et de l’Areg. Cela suggère que les ILC2s migrants de façon spécifique possèdent une empreinte fonctionnelle. Nous confirmons les fonctions des groupes d’ILC2s circulants en utilisant des modèles plus physiologiques mimant des réactions allergiques et des infections parasitaires (stimulation par la papaïne et le succinate). Les migrations vers l’intestin et les poumons jouent un rôle primordial dans l’induction de réponse de type Th2 par sécrétion d’IL-5 et d’IL-13, et à l’initiation de la réparation tissulaire par production d’Areg. De façon intéressante, les ILC2s migrants vers les poumons participent au renouvellement des populations résidentes participant principalement à la production d’Areg. Finalement, nous caractérisons un rôle important du trafic des ILC2s à différents temps suivant l’infection par Nippostrongulus brasiliensis, confirmant la fonction des ILC2s migrantesILC2s are found in mucosal tissues as lung and intestine, in lymph nodes, and in metabolic tissues such as the adipose tissues. They play important role in maintaining or inducing type-2 immune responses as innate equivalent of Th2 lymphocytes. They are activated by alarmins (IL-25 and IL-33) and by external activators (allergens, metabolites and neuromediators). ILC2s are secreting type-2 cytokines to facilitate the activation of other cells and to induce an important repair program. Their activation allows large type of events as diverse as myeloid cells recruitment and activation, mucus production, muscle contractility and tissue repair. They have key role in lung and adipose tissue development and maintain their homeostasis by early responding against parasitic pathogens. Abnormal activation of ILC2s is also participating to chronic diseases.ILCs are mostly considered as resident cells. However, different studies suggested that migration could be important for the maturation of their effector capacities and to correctly target the injured tissue. Circulation and trafficking of ILC subsets is still unclear. No mechanism is yet available to explain the turnover of ILC2s and how they can act in many tissues following stimuli.We found that large numbers of mature and immature ILC2s could be collected in the thoracic duct lymph of mice perfused over several hours, showing that ILC2s are in fact actively circulating through the hemo-lymphatic circuit. Furthermore, circulating mature ILC2s could be separated into three distinct subsets depending on their pattern of receptor and adhesion molecule expression. Cell transfer experiments proved that specific patterns are representative of specific tropism for gut, lung and adipose tissues.To analyse ILC2 behaviour in the context of a type-2 response, we injected IL-25 and IL-33 before lymph collection. IL-33 stimulation largely enhanced the number of circulating ILC2s in the lymph. These different ILC2 tissue targeted subsets responded differently to IL-33. Specifically, gut-trafficking ILC2s were mainly stimulated to proliferate whereas lung and adipose tissue subsets were stimulated to produce IL-13, IL-5 and Areg. This suggests that, in ILC2s, specific tissue targeting is associated with already imprinted functions while transiting through the hemo-lymphatic system. We confirmed these functions of circulating ILC2 subsets in more physiological context by mimicking allergy and helminth infection (stimulation by papain and succinate) where specific migration to lungs and intestine play important roles in mounting the type-2 response by IL-5/IL-13 secretion, and also initiating tissue repair by Areg production. Interestingly, we showed that lung migrating ILC2s participated to resident pool renewal that main function is Areg production. Finally, we characterized important trafficking of ILC2 at different stages of Nippostrongulus brasiliensis infection, confirming the functional relevance of ILC2 trafficking
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Kharabi, Masouleh Schekufe [Verfasser]. "Lipid droplet formation drives pathogenic ILC2 responses in airway inflammation / Schekufe Kharabi Masouleh." Bonn : Universitäts- und Landesbibliothek Bonn, 2020. http://d-nb.info/1218301783/34.
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Schmitt, Pauline. "Rôle de l’IL-33 et des cellules lymphoïdes innées de type 2 dans l’inflammation allergique pulmonaire." Thesis, Toulouse 3, 2019. http://www.theses.fr/2019TOU30283.
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L’exposition aux allergènes environnementaux joue un rôle crucial dans l’exacerbation de maladies allergiques telles que l’asthme. Il est donc important de comprendre pourquoi et comment le système immunitaire réagit aux allergènes. Nos travaux montrent que l’interleukine-33 (IL-33), une cytokine épithéliale de type alarmine jouant un rôle essentiel dans l’inflammation allergique et l’immunité de type 2, détecte l’activité protéolytique associée à une grande variété d’allergènes environnementaux. Lors de l’exposition à ces protéases exogènes, l’IL-33 humaine pleine taille (IL-33FL) est rapidement clivée en formes matures plus courtes et plus actives contenant le domaine cytokine. L’activation de l’IL-33 induit alors la production de cytokines de type 2 (IL-5 et IL-13) par les cellules lymphoïdes innées de type 2 (ILC2s). Les ILC2s sont des cellules résidentes des muqueuses qui contribuent notamment à l’inflammation allergique pulmonaire en induisant une forte éosinophilie et la production de mucus dans les poumons. Nous avons également démontré que la reconstitution de souris déficientes en IL-33 avec l’IL-33FL humaine permet de restaurer l’inflammation pulmonaire (éosinophilie) en réponse à des protéases d’allergènes. Enfin, des anticorps dirigés contre le domaine «capteur» central inhibent le clivage de l’IL-33FL par ces protéases et réduisent l’inflammation allergique des voies respiratoires. Nos résultats révèlent ainsi un mécanisme moléculaire permettant l’induction rapide de l’inflammation allergique de type 2 après une exposition à un allergène, avec des implications importantes pour les maladies allergiques. Nos travaux montrent également qu’il existe une synergie entre l’IL-33 et la cytokine TL1A de la famille du TNF pour induire la sécrétion de grandes quantités d’interleukine-9 (IL-9) par les ILC2s. Des analyses protéomiques globales non biaisées ont en effet révélé que l’IL-9 était la protéine la plus induite dans les ILC2s stimulées par ces deux cytokines. Plus de 99% des ILC2s expriment l’IL-9 rapidement après co-stimulation et de manière transitoire. Cette importante production d’IL-9 est associée à un changement phénotypique caractérisé par une augmentation de l’expression d’IRF4 et de STAT5 et une diminution d’ICOS, de KLRG1 ainsi que du facteur de transcription clé GATA-3 et de ses gènes cibles (IL-9R, ST2). Enfin, l’inhalation d’IL-33 et TL1A chez la souris induit une forte expansion des ILC2s IL-9high pulmonaires. Des expériences de transfert adoptif ont révélé que les ILC2s IL-9high sont de puissantes inductrices de l’inflammation des voies respiratoires in vivo. De plus, cette population est bien présente lors de l’initiation de l’inflammation pulmonaire dans un modèle murin d’allergie et dépend à la fois d’IL-33 et de TL1A. Notre étude met ainsi en évidence un mécanisme moléculaire impliqué dans l’induction d’une production massive d’IL-9 par les ILC2s et suggère un rôle clé de ces cellules dans l’initiation des réponses inflammatoires de type 2 dans les poumonsExposure to environmental allergens plays a crucial role in exacerbating allergic diseases such as asthma. It is therefore important to understand why and how the immune system responds to allergens. Our work shows that interleukin-33 (IL-33), an epithelial alarmin cytokine with a critical role in allergic inflammation and type 2 immunity, detects the proteolytic activity associated with a wide variety of environmental allergens. Upon exposure to these exogenous proteases, full-length human IL-33 (IL-33FL) was rapidly cleaved into shorter and more active mature forms containing the cytokine domain. This activation of IL-33 induced the production of type 2 cytokines (IL-5 and IL-13) by type 2 innate lymphoid cells (ILC2s). ILC2s are resident immune cells contributing to allergic pulmonary inflammation by inducing strong eosinophilia and mucus production in the lungs. We also found that reconstitution of IL-33 deficient mice with human IL-33FL restored pulmonary inflammation (eosinophilia) in response to allergen proteases. Finally, antibodies directed against the central "sensor" domain inhibited the cleavage of IL-33FL by these proteases and reduced allergic inflammation of the airways. Our results thus reveal a molecular mechanism allowing the rapid induction of type 2 allergic inflammation after exposure to allergens, with important implications for allergic diseases. Our study shows a synergy between IL-33 and TL1A, a cytokine from the TNF family, for induction of massive interleukin-9 (IL-9) production by ILC2s. Unbiased global proteomic approach revealed that IL-9 was the most induced protein in ILC2s in response to IL-33/TL1A challenge. Indeed, more than 99% of the co-stimulated ILC2s rapidly expressed IL-9 and this production of IL-9 was transient. IL-9 production was associated with a phenotypic change characterized by an increase of IRF4 and STAT5 expression and a decrease of ICOS, KLRG1 as well as the key transcription factor GATA-3 and its target genes (IL-9R, ST2). Finally, a single inhalation of IL-33 and TL1A in mice induced a strong expansion of pulmonary IL-9high ILC2s and adoptive transfer experiments revealed that IL-9high ILC2s are potent inducers of airway inflammation in vivo. IL-9high ILC2s were induced in a mouse model of asthma during the initiation of allergic airway inflammation, through IL-33- and TL1A-dependant mechanism. Our study thus highlights a molecular mechanism involved in the induction of massive IL-9 production by ILC2s and suggests a key role of these cells in the initiation of inflammatory type 2 responses in the lungs
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Corral, Dan. "Régulation métabolique de la plasticité des ILC2 au cours de l'infection par Mycobacterium tuberculosis." Thesis, Toulouse 3, 2020. http://www.theses.fr/2020TOU30177.
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Les muqueuses hébergent des cellules immunitaires résidentes qui jouent un double rôle : elles contribuent au maintien de l'intégrité tissulaire et à la protection du tissu lors d'infection par des pathogènes. Parmi celles-ci, les cellules lymphoïdes innées (ILC) sont des actrices clés de l'homéostasie tissulaire et de la réponse immunitaire. Les dernières années ont révélé l'existence de différents types d'ILC, classées selon leur similarité avec les lymphocytes T au niveau de l'expression de facteurs de transcription et de leurs fonctions effectrices. Ainsi, on retrouve les ILC1, les ILC2 et les ILC3 qui forment les "contreparties" innées des cellules T CD4+ de type Th1, Th2 et Th17 respectivement, ainsi que les NK, contrepartie des lymphocytes cytotoxiques CD8+. De façon similaire aux sous-types de Th, les différents types d'ILC ont été associés à divers pathologies. Au sein du tissu pulmonaire, les ILC2 constituent le sous-type quantitativement majoritaire d'ILC chez les rongeurs et leur rôle a notamment été caractérisé dans des pathologies de type 2 (asthme, allergies, infections parasitaires). Le poumon représente le site d'entrée pour de nombreux agents infectieux, mais le rôle des ILC2 lors des infections pulmonaires d'origine bactérienne reste peu exploré. Au cours de ma thèse, je me suis intéressé au rôle des ILC, et particulièrement des ILC2, dans le modèle murin d'infection par Mycobacterium tuberculosis (Mtb), l'agent étiologique de la tuberculose (TB). L'infection par Mtb induit principalement une réponse immunitaire de type 1, l'IFNƴ produit permettant l'activation des fonctions microbicides des macrophages infectés. Néanmoins, ce mécanisme dominant de l'immunité antituberculeuse peine à prédire l'issue de l'infection et la vision actuelle est que d'autres acteurs cellulaires contribuent probablement de façon importante à la protection. Sur la base de leur présence dans le poumon à l'entrée du pathogène et de la diversité de leur potentiel effecteur antimicrobien et protecteur du tissu, nous avons donc émis l'hypothèse que les ILC pouvaient être activées et participer à la réponse immunitaire antituberculeuse. Dans le modèle murin de la tuberculose, nous avons pu montrer que les ILC étaient diversement régulées au cours de l'infection : alors que les ILC1 et les ILC3 se développent et s'activent, les ILC2 se contractent et sont inhibées. De façon intéressante, l'inhibition des ILC2 est associée à un mécanisme de plasticité caractérisé par la perte de marqueurs d'ILC2 tels que GATA3, ST2, Arg1 et IL-5 et l'acquisition de marqueurs caractéristiques des ILC1 comme T-bet, IL-18Ra, CD49a et IFNƴ. Différents stades de la plasticité des ILC2 ont été identifiés sur la base de l'expression de CD49a et d'IL-18Ra conduisant à la formation de cellules ILC1-like, présentant un potentiel protecteur au cours de l'infection.[...]Mucosal tissues harbor resident immune cells that play a dual role in maintaining both tissue integrity and protection against infection by pathogens. Among these, innate lymphoid cells (ILCs) are key players in tissue homeostasis and immune response. The last few years have revealed the existence of different types of ILCs, classified according to their similarity to T cells based on expression of dedicated transcription factors and the execution of specific effector functions. These include ILC1, ILC2 and ILC3, which form the innate counterparts of CD4+ T cells of the Th1, Th2 and Th17 types respectively, as well as NK for CD8+ cytotoxic lymphocytes. Similar to the associated Th subtypes, the different types of ILC have been implicated in various diseases. Within lung tissue, ILC2 is the quantitatively dominant subtype of ILC and its role has been characterized in particular in type 2 pathologies (asthma, allergies, parasitic infections). The lung is the site of entry for many infectious agents: yet the role of ILC2 in bacterial lung infections remains poorly explored. During my thesis, I was interested in the role of ILC, particularly ILC2, in the mouse model of Mycobacterium tuberculosis (Mtb) infection, the etiological agent of tuberculosis. Infection with Mtb typically induces a type 1 immune response: IFNƴ production allows the activation of the microbicidal functions of infected macrophages. Nevertheless, this dominant mechanism of TB immunity barely predicts the outcome of infection, and the current view is that other cellular actors are likely to contribute significantly to protection. Based on their presence in the lung at the entry of the pathogen and the diversity of their antimicrobial and tissue-protective effector potential, I hypothesized that ILC could be activated and participate in the antituberculous immune response. In the mouse model of tuberculosis, I could show that ILC are differentially regulated during infection: while ILC1 and ILC3 expand and become activated, ILC2 contract and become functionally inhibited. Interestingly, inhibition of ILC2 is associated with a plasticity mechanism characterized by the loss of ILC2 markers, such as GATA3, ST2, Arg1 and IL-5, together with the acquisition of ILC1 characteristic markers, such as T-bet, IL-18Ra, CD49a and IFNƴ. Different stages of ILC2 plasticity were identified based on the expression of CD49a and IL-18Ra, leading to the formation of ILC1-like cells, which display a protective potential during infection. In this infectious model, as well as through the development of an easier model of plasticity based on the administration of cytokines, we showed that IFNƴ, originating from ILC1 and NK cells, as well as the expression of the transcription factor STAT1, were essential components for the generation of ILC1-like cells. With the aim to identify the molecular mechanisms governing this plasticity, we hypothesized that the ILC2-to-ILC1-like cell plasticity was associated with a marked metabolic change. [...]
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Wu, Ching-Lien. "Study of the immune checkpoint HLA-G/ILT2 : soluble receptors, inhibition of iNKT cells." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC188.
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HLA-G est une molécule checkpoint immunologique tolérogène connue pour son rôle dans la tolerance materno-foetale, puis comme mécanisme d’échappement immunitaire des tumeurs. HLA-G exerce ses fonctions via avec deux récepteurs inhibiteurs principaux, ILT2 et ILT4, différentiellement exprimés par les effecteurs immunitaires. L’expression de HLA-G par les tumeurs solides est associée à de plus hauts grades et à un pronostic plus sévère. Pour ces raisons, HLA-G et ses récepteurs pourraient constituer de bonnes cibles immuno-thérapeutiques en oncologie.Ce travail de doctorat a porté sur 1) l’existence et la pertinence clinique des formes solubles de ILT2 et ILT4, ainsi que sur 2) la fonction de la molécule HLA-G sur les cellules iNKT.1) Existence et pertinence clinique des formes solubles de ILT2 et ILT4 : nous avons tout d’abord développé un test Luminex de dosage des formes solubles de ILT2 et ILT4. Ce test est spécifique des molecules sILT2 et sILT4 libres (i.e. non complexées à HLA-G) et nous a permis d’établir que des formes solubles de ILT2 existaient in vivo et circulaient dans le sang périphérique. Cependant, nos cohortes ne nous ont pas permis de détecter des formes solubles d’ILT4 circulantes. Nous avons mesuré les niveaux de sILT2 et sILT4 dans le sang périphérique de (i) patients atteints de cancers de type B-CLL, lymphome B et cancer de vessie non-infiltrant le muscle, (ii) patients atteints de dégénérescence maculaire liée à l’âge, et (iii) patients atteints de troubles bipolaires. Malgré des niveaux de sILT2 plus élevés chez certains patients atteints de cancer, nos études n’ont pas permis d’atteindre de pertinence statistique.2) Fonctions de la molécule HLA-G sur les cellules iNKT : les cellules iNKT ont été décrites comme inductrices de réponses anti-tumorales dans des modèles expérimentaux murins et font l’objet d’études à visée thérapeutique comme outils immuno-thérapeutiques soit par elles-mêmes, soit comme adjuvants. Les cellules iNKT sont une sous-population minoritaire de lymphocytes T qui reconnaissent des lipides présentés dans le context de CD1d. HLA-G étant communément exprimée par les tumeurs humaines, il est donc important de caractériser sa function sur les cellules iNKT. Nos travaux démontrent que les cellules iNKT n’expriment que très peu ILT2, mais que cette expression est induite par l’activation. Nous avons aussi démontré que HLA-G inhibe la production cytokinique des cellules iNKT, soit directement via ILT2, soit indirectement par l’intermédiaire de cellules myeloides tolérogènes. Ces résultats démontrent l’intérêt d’étudier HLA-G et ses récepteurs ILT2 et ILT4 concommitamment et suggèrent que le blocage du checkpoint immunologique HLA-G/ILT2 constitue une stratégie adjuvante valide afin d’améliorer l’efficacité des immunothérapies visant des tumeurs exprimant HLA-GHLA-G is a tolerogenic immune checkpoint molecule, first known for its role in maternal-fetal tolerance and now well described as an immune escape mechanism for tumors. HLA-G exerts its functions through interaction with two main inhibitory receptors, ILT2 and ILT4 that are differentially expressed by immune effector cells. HLA-G expression by solid tumors is associated with higher tumor grade and a worse prognosis. Thus, HLA-G and its receptors are currently being investigated as therapeutic targets in the context of oncology.In this PhD project, we focused on 1) the existence and clinical relevance of soluble ILT2 and ILT4 molecules, and 2) on the impact of HLA-G on the function of iNKT cells. 1) Existence and clinical relevance of soluble ILT2 and ILT4 molecules: we developed a Luminex-based assay in order to measure the concentrations of sILT2 and sILT4. This assay was able to detect free sILT molecules, i.e. not complexed with HLA-G, and allowed us to establish that sILT2 molecules did circulate in the peripheral blood. sILT4 molecules were not detected in this tissue. We measured sILT2 and sILT4 levels in the peripheral blood of (i) cancer patients with B-CLL, B-cell lymphoma, and non-muscle-invasive bladder cancer, (ii) patients with macular degeneration, and (iii) patients with bipolar disorders. Even though higher sILT2 levels could be observed in some cancer patients, no significance was reached. 2) Impact of HLA-G on the function of iNKT cells: iNKT cells were reported to be potent inducers of anti-tumor responses in animal models and they are currently investigated as anti-tumor therapeutic tools either as stand-alone or adjuvant therapy. iNKT cells are a small subset of T cells that recognize lipids presented in the context of the MHC class I-like molecule, CD1d. Because HLA-G is commonly expressed by human tumors, it was therefore important to decipher its function on iNKT cells. Working with murine and human cells, we established that human iNKT cells do not constitutively express ILT2 but upregulate it upon stimulation. We also demonstrated that HLA-G inhibited the cytokine secretion function of iNKT cells, directly through this receptor and also indirectly through tolerogenic antigen-presenting cell.These data highlight the interest of studying ILT2 concommitantly to HLA-G and suggest that blocking the HLA-G/ILT2 checkpoint should constitute a valid adjuvant strategy to improve anti-tumor immune therapies for HLA-G-expressing tumors
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Hwang, You Yi Leon. "Investigating the roles and type 2 cytokine profiles of ILC2 using novel mouse models." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709227.
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Albaker, Awatif. "Mutational Analysis to Define the Functional Role of the Third Intracellular Loop of D1-Class Dopaminergic Receptors." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/35063.
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The third intracellular loop (IL3) and cytoplasmic tail (CT), which are the most divergent regions between human D1-class dopaminergic receptors (hD1R and hD5R), have been implicated in modulating their subtype-specific functional phenotypes. The importance of the IL3 for Guanine nucleotide-binding protein (G-protein) coupling and specificity has long been acknowledged in the G-protein-coupled receptor (GPCR) field. However, the exact role the central region of the IL3, notably the N- and C-terminal moieties, plays in GPCR receptor functionality remains unclear. Studies in our laboratory indicated that the IL3/N-terminal moiety of hD1-class receptors appears to be critical for facilitating agonist-independent and dependent activation of hD1R and hD5R. Furthermore, the IL3/C-terminal portion of hD1-class receptors constrains the receptor in the inactive state and reduces receptor affinity for agonists and G-protein coupling. I put forward the following hypothesis: 1. The functional properties of hD1-class receptors are regulated via a molecular micro-switch present within the IL3 central region modulating the functional properties of the receptor distinctly, 2. The functional differences between D1R and D5R require structural elements from both N- and C-terminal halves of the IL3 central region, and 3. The molecular interplay between the N- and C-terminal halves of the IL3 central region is dependent on the amino acid chain length and content. Herein, I have employed site-directed mutagenesis, and alanine replacement approaches to analyze comprehensively the structural determinants within the N- and C-terminal moieties of the IL3 central region that regulate ligand binding and G-protein coupling properties of hD1-class receptors. Moreover, my Ph.D. research aimed to pinpoint whether the IL3 length and/or structural motif(s) regulate ligand binding and activation properties of hD1R and hD5R. The results of my study highlight the importance of structural elements from both the proximal and distal segments of the IL3/central region of hD1-class receptors for the ligand binding and receptor activation status. Additionally, my results underline the significance of preserving the length of the IL3 regardless of the amino acid content. This study also shows the pivotal role played by a phenylalanine residue, F2646.27, in the signaling properties of hD1R. Notably, mutating F2646.27 leads to a mutant hD1R with characteristics resembling those of constitutively active mutant GPCRs. Unraveling the amino acid/amino acids constraining the receptor in the inactive state will perhaps provide an attractive target for drug design. Future work aims at developing drugs that particularly bind to the intracellular face of hD1R and improving selectivity towards hD1R may prove useful in limiting the side effects associated with the conventional therapy of brain disorders such as in the case of L-DOPA induced dyskinesia (LID) seen in individuals suffering from Parkinson’s disease.
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Heinrup, Rebecka. "Evaluation of isobutanol tolerance and gene expression in four different Saccharomyces cerevisiae strains for the development of bio-butanol production." Thesis, Linköpings universitet, Biologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-132314.
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Today, most transportation fuels are derived from crude oil. However, fossil fuels are limited resources and contribute to climate change, and are therefore not considered as sustainable. Biofuels are highly relevant candidates for replacing fossil fuels and research has gone into butanol as a biofuel. It has a high energy density, is less hygroscopic and can be blended up to 85% with gasoline. The yeast Saccharomyces cerevisiae is considered a good host for bio- butanol production; it produces small amounts of isobutanol naturally through the Ehrlich pathway, is easy to manipulate genetically and can therefore be engineered to produce higher titres of butanol. End-product toxicity, however, is a problem that needs to be solved to make butanol production in S. cerevisiae more effective, since the organism cannot tolerate higher concentrations of butanol than 2%. Four different S. cerevisiae strains were cultivated in 1.5%, 2%, 3% and 4% isobutanol by spot tests and in liquid media to evaluate their tolerance. Gene expression was measured for genes RPN4, RTG1 and ILV2 to examine their up-regulation and relevance in butanol tolerance. S. cerevisiae strain Saflager 34/70 was determined as the most tolerant strain and was able to grow in 2% liquid isobutanol and 3% isobutanol on agar plates. A three-fold up-regulation of RPN4, a transcription factor involved in the regulation of proteasome gene expression, was observed. These results contribute to the progress of genetic engineering of butanol host organisms, which is needed to create a more effective production of butanol as a biofuel.
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Taylor, Patricia R. "J-LEAPS VACCINES ARE SUFFICIENT TO ACTIVATE AND DIRECT AN IMMUNE RESPONSE THROUGH DENDRITIC CELLS." Kent State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=kent1278684731.
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Lownik, Joseph C. "The Role of ADAM10 and ADAM17 in Humoral and Type 2 Immunity." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5680.
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The proper regulation of inducible costimulator (ICOS) and its ligand (ICOSL) have been shown to be essential for maintaining immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant antibody production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that ADAM10 is the primary physiological sheddase of ICOSL in both mouse and human. Using an in vivo system in which ADAM10 is deleted only on B cells (ADAM10B-/-), elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wildtype (WT) mice, interaction of ICOSL/ICOS results in ADAM10 induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper (TFH) development and Th2 polarization, seen in a house dust mite exposure model. In addition, enhanced Th1 and Th1 immune responses are seen in experimental allergic encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and at least partially rescues both TFH numbers and the abnormal antibody production previously reported in these mice. Overall, we propose a novel regulation of the ICOS:ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL as well as indirectly regulating ICOS, thus controlling ICOS:ICOSL-dependent responses.
Additionally, we report a specific role for the metalloprotease ADAM10 on B cells in regulating both ICOSL and ICOS in a mouse model of increased humoral immunity using mir146a-/- mice and a model of lymphoproliferative disease using the well characterized lpr model. B6lpr mice lacking ADAM10 on B cells (A10Blpr) have decreased nodal proliferation and T cell accumulation compared to control B6lpr mice. Additionally, A10Blpr mice have a drastic reduction in autoimmune anti-dsDNA antibody production. In line with this, we found a significant reduction in follicular helper T cells (TFH) and germinal center (GC) B cells in these mice. We also show that lymphoproliferation in this model is closely tied to elevated ICOS levels and decreased ICOSL levels. Overall, our data not only shows a role of B cell ADAM10 in controlling autoimmunity, but also increases our understanding of the regulation of ICOS and ICOSL in the context of autoimmunity.
Additionally, we found that ADAM17 is important for marginal zone (MZ) B cell development as well as responses to T-independent type 2 (TI2) immunizations. Mice which lack ADAM17 on B cells (A17B) have decreased MZ B cell numbers but have increased levels of antigen specific antibodies in response to TI2 Immunizations. ADAM17 also regulates the level of several surface molecules on plasma cells and MZ B cells necessary for their function and survival.
We also show a role for ADAM17 in ILC2 responsiveness to IL-33. In vivo, mice that lack ADAM17 specifically on ILC2s (ADAM17ILC2-/-) exhibit decreased ILC2 expansion in response to intranasal IL-33 as well as Nippostrongylus brasiliensis (Nb) infection. However, ADAM17ILC2-/- mice have normal ILC2 numbers in a naïve state, suggesting this defect in ILC2 function is limited to cell activation. In vitro, ADAM17 inhibited ILC2s have an increased level of apoptosis and less IL-13 production in response to IL-33 compared to vehicle treated ILC2s. The defect in cytokine production following ADAM17 inhibition is not observed in response to IL-25 stimulation, suggesting this defect is limited to IL-33 stimulation Mechanistically, ADAM17 inhibition in ILC2s specifically causes a defect in IL-33 mediated ERK activation, potentially explaining the defective survival and IL-13 production following ADAM17 inhibition in these cells. Additionally, ADAM17 regulates the level of surface IL1R2 which may affect IL-33 signaling in ILC2s.
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D'Anca, M. "IDENTIFICATION AND VALIDATION OF CRITICAL 1Q21 'ACHILLES HEEL' VULNERABILITIES OF MULTIPLE MYELOMA." Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/362028.
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Multiple Myeloma (MM) is malignancy of terminally differentiated plasma cells characterized by a marked heterogeneity of genetic lesions and clinical course. Despite significant efforts towards the development of risk stratification strategies for patients with Multiple Myeloma (MM), we are still limited in our capacity to molecularly predict the natural history of these patients. Recent molecular analyses have illuminated many aspects of the pathogenesis of this heterogeneous disease, although there remains an elemental view of the compendium of genetic elements driving MM initiation and progression and how such genetic alterations functionally contribute to specific aspects of disease pathobiology, prognosis and treatment responses. Indeed, despite considerable progress in the management of MM patients, many studies have shown that some genetic alterations especially t(4;14) translocation, loss of the short arm of chromosome 17, loss of the long arm of chromosome 13 and amplification of chromosome 1q21 remain associated with a poor outcome and represent independent adverse predictors of shorter progression free survival (PFS) and overall survival (OS). The 1q21 amplicon is among the most frequent chromosomal aberrations in patients with MM (about 40% of de novo MM) and is considered a highly poor-risk genetic feature correlated with disease progression and drug resistance; it spans approximately a region of 10-15 Mb containing a large number of possible candidate genes. To date the relevant genes on 1q21 remain unclear and the absence of focal amplifications involving this region strongly suggests that more than a single candidate may represent the driver event responsible for poor outcome of this group of MM patients. Thus, the identification of critical 1q21 ‘Achilles heel’ vulnerabilities may yield a comprehensive catalog of the potential therapeutic targets for these high risk MM and provide a rationale for patient stratification. In an effort to accomplish this goal, we first identified a high-priority list of 78 copy number-driven 1q21 MM-relevant genes. Then, we have designed a high-throughput systematic shRNA screen approach in vitro to identify 1q21 genes whose loss of function results in selective death and/or growth inhibition of MM cells carrying the 1q21 amplification. After excluding shRNAs that display citotoxic activity regardless 1q21 amplification, we defined 1q21 “Achilles heel” vulnerabilities as shRNA target genes whose down-regulation decreases substantially the percentage of GFP-positive MM cells with 1q21 amplification over a time of 7 days based on a GFP-competion assay. This assay provided a list of candidate genes implicated in survival or proliferation of MM cells with 1q21 amplification; MCL1, UBAP2L, INTS3, LASS2, KRTCAP2, and ILF2. By targeting these six genes we performed secondary validation experiments in JJN3 and H929 MM cell lines, carrying 4 copies of 1q21 amplicon. The results of this secondary validation confirmed that the down-regulation of these genes caused an important decrease of proliferation and increase of apoptosis as well as growth cycle arrest. Further GEP analysis and clinical outcome studies revealed that only UBAP2L and ILF2 showed a significant prognostic value but in vivo validation studies on NOD-SCID mice identified only ILF2 correlated with in vivo survival. So our studies focused to investigate the role of ILF2 in 1q21 amplified MM.
Nuclear Factor 45 (NF45) or ILF-2 is widely expressed in normal tissue with a predominant nuclear distribution. NF45/ILF2 associates with NF90/NF110 (ILF3) interacting with DNA and RNA. ILF2 and ILF3 contribute to gene regulation at different levels, transcription, splicing, nuclear exporting, but they are also involved in other important processes like mitotic control and DNA break repair.
Down-regulation of ILF2 in MM cells with 1q21 amplification resulted in multinucleated phenotypes and abnormal nuclear morphologies that were associated with a significant accumulation of γH2AX foci and DNA damage response activation, increased sensitivity to Melphalan, DNA damaging agent, and impaired activation of DNA repair pathways. Experiments of immunoprecipitation combined with mass spectometry showed that ILF2 interacts with numerous RNA binding proteins directly implicated in DNA repair or regulation of DNA damage response by modulating alternative splicing and stability of specific pre-mRNAs. Accordingly, RNA-sequencing analysis of ILF2-depleted MM cells, when compared to cells carrying scrambled shRNAs, identified specific changes in RNA splicing patterns before and after treatment with Melphalan.
Thus, our findings have raised a new tight correlation between 1q21 amplification and DNA damage response. We identified ILF2 as a key driver of this interaction, and our findings support the development of strategies designed to modulate ILF2 expression in patients with high-risk MM carrying 1q21 amplification providing personalized therapies for patients who do not benefit from recent treatment improvements.
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Pascal, Maud. "Innate Lymphoid Cells under Neuronal Control in the Small Intestine : vasoactive Intestinal Peptide potentiates ILC2 and ILC3 functions." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS318.
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L’intestin est une vaste surface de l’organisme constamment exposée aux substances ingérées et aux nombreux micro-organismes qui colonisent sa muqueuse. Afin d’assurer le maintien de son intégrité, plusieurs dispositifs de reconnaissance et de défense impliquent cellules immunitaires et neurones, faisant de lui un organe de choix pour l’étude des interactions neuroimmunes. La compréhension des éléments assurant un fonctionnement coordonné des Systèmes immunitaire (SI) et Nerveux (SN) dans l’intestin reste cependant partielle. Ce travail a porté sur l’étude des mécanismes régissant le fonctionnement intégré des SN et SI de l’intestin suite à une prise alimentaire. Nous démontrons que la libération de Peptide Intestinal Vasoactif (VIP) suite à une prise alimentaire impacte la fonction de cellules clés dans l’organisation de l’immunité intestinale, les cellules lymphoïde innées (ILC). Pour la première fois, on démontre qu’un neuropeptide modifie de manière anticipée la biologie des ILC2 et des ILC3 pour potentialiser l’effet de cytokines inductrices caractéristiques des immunités de type 2 ou de type 3, permettant une activation rapide et conséquente des ILC. Ce travail complexifie le réseau de régulation du fonctionnement des ILC et dévoile un nouveau rôle du VIP dans le maintien de l’homéostasie intestinale via sa capacité à anticiper et potentialiser les réponses immune de manière intégrée. La compréhension de ce nouveau mécanisme d’interactions neuroimmunes dans l’intestin ouvre la voie vers le développement de nouvelles stratégies thérapeutiques basées sur les propriétés du VIP pour prévenir et traiter des maladies infectieuses et inflammatoires de l’intestinThe intestine represents an extremely wide interface constantly exposed to substances that we ingest and to numerous micro-organisms that colonize its mucosae. Several mechanisms of recognition and defense involving both immune cells and neurons exist to ensure protection of the gut, setting the gut as a paradigm for neuroimmune interactions. However, how the nervous and immune systems coordinate and synchronize their action in the gut remain unclear. In this thesis, I aimed to elucidate the mechanisms underlying one type of neuroimmune communication occurring in the gut, during a physiological process: feeding. In this context, I demonstrated that the food-induced release of the Vasoactive Intestinal Peptide (VIP) impacts the function of the recently discovered “gatekeepers” of the gut immune system, Innate Lymphoid Cells (ILCs). For the first time, I showed that a neuropeptide induces an anticipatory priming of both ILC2 and ILC3, which could potentiate the effect of the canonical type 2 and type 3 inducer cytokines to lead a rapid and strong activation of ILCs. This work provides new insights in the highly complex regulatory network of ILCs and uncovers a new role for VIP in maintaining gut homeostasis through its ability to prime and eventually boost immune responses in an integrated and context dependent manner. The understanding of the neuroimmune interplay involving VIP in the small intestine opens the path toward the development of new therapeutic strategies based on VIP properties to treat infectious and inflammatory diseases of the gastrointestinal tract
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Penafuerte, Diaz Claudia. "IL2-based fusion proteins as new bi-functional biopharmaceuticals for the therapy of cancer." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103578.
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The murine fusion protein between Granulocyte macrophage Colony Stimulating Factor (GM-CSF) and Interleukin-2 (IL2, aka GIFT2) display novel immunological properties compared to both cytokines in combination such as greater melanoma site recruitment of macrophages and functional NK cells. Consequently, GIFT2 prevent tumor formation in mice implanted with genetically modified B16 melanoma cells. In the Chapter 2 of my thesis, I evaluated the bystander effect of the murine GIFT2 in vivo to induce an effective antitumor response against non-genetically modified B16 cells present in the tumor site. GIFT2 bystander effect on non modified B16 cells was mediated by recruited NK cells in the tumor site. However, the immune bystander effect was completely lost as tumor burden increase, which correlated with a sharp reduction in the number of tumor-infiltrating NK cells. I identified that active TGF is the main tumor-derived factor that downregulated IL-2R expression and IFNg secretion by NK cells, and therefore attenuated GIFT2-dependent bystander effect. We demonstrated that in vivo blockade of B16-derived TGF significantly improved the immune bystander effect arising from GIFT2. Based on the potent immunostimulatory properties of the murine GIFT2 on NK cells, in the chapter 3 I developed, characterized and evaluated the human ortholog of GIFT2, which may serve as a mean to generate oncolytic NK cells for cell-based therapy of cancer. The human GIFT2 induces robust NK cell activation ex vivo with significant secretion of pro-inflammatory cytokines, chemokines and upregulate the expression of activation markers and receptors on NK cells. This phenotype correlates with significantly greater cytotoxicity against tumor cells. At the molecular level, the human GIFT2 leads to a potent activation of Jak/STAT signaling pathway downstream of IL-2 receptor. In conclusion, hGIFT2 fusokine possesses unique biochemical properties and constitutes a novel and potent tool for ex vivo NK cell activation and maturation. Based on our results, cancer gene immunotherapy of pre-established tumors will be enhanced by blockade of active TGF. To antagonize TGF dependent effects in tandem with a pro-inflammatory immune stimulus, I generate of a new chimeric protein borne of the fusion of IL-2 and the soluble extracellular domain of TGF-receptor II (aka FIST). FIST acts as a decoy receptor trapping active TGF-in solution and directly interacts with IL-2-responsive cells, inducing a distinctive hyperactivation of STAT1 downstream of IL-2 receptor, which in turn promotes SMAD7 overexpression. STAT1 hyperactivation further induces significant secretion of CXCL10, upregulates T-Bet and T-Bet target gene expression in NK cells. The synergism of TGFblockade coupled to IL-2(R)-dependent STAT1 hyperagonism leads to potent immune activation contemporaneous to a dominant NK cell-dependent antiangiogenic effect in the B16 murine model of melanoma. Consequently, FIST prevent tumor formation not only in immunocompetent mice but also in several immunodeficient mice, whereas mice with NK defective functions such as nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice and Rag2/c KO mice developed tumors. In the chapter 5, I generate and characterize FIST-stimulated B cells. FIST-stimulated B cells upregulate co-stimulatory molecules, activation markers and MHC class II molecule expression, which is also supported by robust hyperactivation of Jak/STAT signaling pathway. FIST-stimulated B cells act as effective APC that induce the activation and cell proliferation of antigen-specific CD4+ and CD8+ T cells. Interestingly, FIST-stimulated B cells confer complete protective immunity to EG.7 tumor challenge in vivo. Therefore, FIST can also be used as stimulator to generate B cells with APC features useful for the cell-based therapy of cancer. In conclusion, these bi-functional chimeric proteins are potential biopharmaceuticals for the therapy of cancer.La protéine de fusion comprenant le facteur stimulant les colonies des granulocytes-macrophages murins (GM-CSF) et l'interleukine-2 (IL2), GIFT2. GIFT2 possède de nouvelles propriétés immunologiques par rapport à l'utilisation des deux cytokines, telles que le recrutement d'un plus grand nombre de macrophages et de cellules NK dans un mélanome. Par conséquent, GIFT2 empêche la formation de tumeurs chez la souris implantée avec des cellules de mélanome B16. Dans le chapitre 2, j'ai évalué la capacité de GIFT2 à induire une réponse anti-tumorale in vivo contre des cellules B16 non-modifiées. J'ai remarqué que GIFT2 induit un effet spectateur sur les cellules B16 non-modifiées, et que cet effet est médié par les cellules NK. Toutefois, cet effet spectateur immunitaire se perd lorsque le nombre total de cellules B16 passe de 104 à 106. Avec ce plus grand nombre de cellules B16, j'ai observé une réduction substantielle du nombre de cellules NK infiltrants. J'ai déterminé que le facteur principal produit par la tumeur qui supprimait l'effet spectateur de GIFT2 est le TGF actif. J'ai observé que le TGF actif a diminué l'expression du récepteur de l'IL-2 (IL-2R) ainsi que la sécrétion de l'IFNg par les cellules NK. J'ai démontré que lorsque la sécrétion de TGF par les cellules B16 est inhibée, l'effet spectateur de GIFT2 est considérablement amélioré. Due aux propriétés immunostimulantes de la protéine de fusion GIFT2 murine, j'ai développé et évalué l'orthologue humain de GIFT2. Ceci est décris dans le chapitre 3. La GIFT2 humaine peut servir comme un moyen de générer des cellules NK oncolytiques pour la thérapie cellulaire du cancer. J'ai remarqué que la GIFT2 humaine induit une activation robuste de cellules NK ex vivo avec une sécrétion significative de cytokines/chemokines pro-inflammatoires et une expression augmentée de marqueurs d'activation de cellules NK. Ce phénotype est corrélé à une plus grande cytotoxicité contre les cellules tumorales. Au niveau moléculaire, la GIFT2 humaine mène à une activation puissante de la voie de signalisation Jak/STAT. Suivant ces résultats, j'ai proposé que l'inhibition du TGF actif pourrait améliorer l'immunothérapie génique d'un cancer existant. Dans le chapitre 4, je décris la production d'une nouvelle protéine de fusion entre l'IL-2 et le domaine extracellulaire du récepteur II de TGF (TGFRII soluble). Cette protéine chimérique appelé FIST a été générée pour contrarier les effets du TGF et aussi pour agir comme stimulus immunitaire pro-inflammatoire. J'ai observé que FIST agit comme récepteur du TGF actif qui se trouve en solution, et aussi interagit directement avec les cellules IL-2-sensibles. Ceci induit une hyperactivation de STAT1 en aval du récepteur de l'IL-2, ce qui donne lieu à une surexpression de SMAD7. De plus, l'hyperactivation de STAT1 induit une sécrétion significative de CXCL10, et augmente l'expression de T-bet et des gènes cibles de T-bet dans les cellules NK. Par conséquent, FIST empêche la formation de tumeurs non seulement chez la souris immunocompétente, mais aussi chez différentes souris immunodéficientes. Par contre, les souris avec fonction NK défectueuse, telles que les souris diabétiques non obèses présentant une immunodéficience combinée sévère (NOD-SCID) et les souris Rag2/c-/- ont développé des tumeurs. Tel que décrit dans le chapitre 5, j'ai générer et caractériser les cellules B stimulées par FIST. Les cellules B stimulées par la protéine FIST agissent comme des cellules présentatrices d'antigènes (APC) efficaces qui induisent l'activation et la prolifération de cellules T CD4+ et CD8+ antigène-spécifiques. Ce qui est aussi très intéressant est que les cellules B stimulées par FIST confèrent une immunité protectrice complète contre le "challenge" de la tumeur EG7. En conclusion, ces nouvelles protéines chimères bi-fonctionnelles sont des produits biopharmaceutiques potentiels pour le traitement du cancer.
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LORU, FABRICE. "Analyse des constituants de buxus sempervirens et de leurs proprietes anti-vih et anti-il2." Nice, 1999. http://www.theses.fr/1999NICE5309.
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Ce travail concerne l'analyse de la preparation vegetale de buxus sempervirens administree aux patients dans le cadre d'un essai pilote phase i/ii, mis en place par l'equipe du professeur dellamonica en juin 1992 a l'hopital de nice. Nous avons mis au point une premiere methodologie de fractionnement impliquant un procede d'extraction suivi d'un traitement par chromatographie liquide qui nous a permis d'obtenir une fraction presentant in vitro a la fois une activite antivirale anti-vih et une activite inhibitrice de la production d'il2 interessante. Cependant, nos essais de reproductibilite n'ont pas abouti. Des essais de traitement, effectues en collaboration avec les laboratoires arkopharma indiquent une augmentation de l'activite biologique correlee avec le pourcentage d'alcaloides dans les extraits. Aussi, nous avons prepare un extrait d'alcaloides totaux que nous avons fractionne suivant une methodologie basee sur la difference de basicite des alcaloides en realisant des extractions a differents ph (8-7-6-5-4-3-2). Nous avons ainsi obtenu des fractions constantes dans leurs activites biologiques : faible activite anti-vih, activite inhibitrice notable de la production d'il2. La chromatographie liquide sur gel de silice ou d'alumine de ces fractions nous a permis d'isoler trois alcaloides deja decrits (la (-) 16--hydroxybuxaminone, la buxamine e et le buxaminol e) et quatre alcaloides originaux dont nous avons elucide la structure. Ces composes presentent un squelette 9,10-seco-buxa-9(11), 10(19)-diene, aussi nous les avons nommes : le n 2 0-formylbuxaminol e, le o 1 6-syringoylbuxaminol e, la n 2 0-acetylbuxamine e et la n 2 0-acetylbuxamine g. Dans le but de definir la composition chimique des extraits d'alcaloides totaux de buxus sempervirens, nous avons mis au point une methode d'analyse par couplage clhp-ms qui nous a conduit a l'identification de quinze constituants d'extraits d'alcaloides totaux, a la detection de tous les composants de ces extraits, a l'obtention de spectres uv et a la determination de masse moleculaire de chacun des solutes elues en une seule analyse. Ces travaux constituent la premiere etude de la composition chimique des alcaloides totaux de buxus sempervirens par chromatographie et permettent a la fois d'etablir des profils chromatographiques et de determiner les constituants des extraits a analyser.
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Madouri, Fahima. "Asthme allergique induit par un allergène d’acarien, House Dust Mite (HDM) : rôles de la caspase-1 et de la protéine kinase C thêta (PKC-θ)." Thesis, Orléans, 2014. http://www.theses.fr/2014ORLE2055/document.
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Des études menées au laboratoire avaient démontré un rôle critique de l’inflammasome NLRP3 dans l’asthme allergique en réponse à l’ovalbumine en absence d’adjuvant. Mes travaux de thèse ont porté sur le rôle de NLRP3 et de la caspase-1 dans un modèle murin d’inflammation pulmonaire induite par l’allergène d’acarien HDM. Nous avons montré un rôle régulateur de la caspase-1 dépendant de l’inflammasome NLRP3 et la molécule adaptatrice ASC mais pas de l’inflammasome NLRC4. Cette régulation de la réponse allergique se caractérise par une augmentation de l’infiltration des éosinophiles, de l’hyperréactivité bronchique et de la production des cytokines de type Th2 telles que l’IL-4, l’IL-5, l’IL-13 et l’IL-33 dans les poumons. Nous avons montré que les mécanismes responsables de cette régulation sont associés à l’IL-33 produite par les macrophages et que la neutralisation de l’IL-33 par administration locale de la protéine de fusion au récepteur ST2 (muST2-Fc) atténue les caractéristiques de l’asthme allergique. Ces résultats suggèrent que l’activation de la caspase-1 réduit la production d’IL-33 in vivo et régule ainsi la réponse l’inflammation pulmonaire induite par HDM et la réponse Th2. D’autre part, nous nous sommes intéressés au rôle de la Protéine Kinase C thêta (PKC-θ) dans ce même modèle d’inflammation pulmonaire. Nous avons démontré que PKC-θ joue non seulement un rôle protecteur dans l’asthme allergique mais également un rôle critique pour la prolifération et l’activation des cellules lymphoïdes innées (ILC2). D’autre part, l’inhibition de PKC-θ in vivo par administration orale de son inhibiteur spécifique C20 (BIX02656) atténue l’inflammation pulmonaire et la production d’IL-5 et d’IL-13. Nous suggérons que PKC-θ est impliquée dans la différenciation des Th2 et des ILC2 via un mécanisme dépendant des facteurs de transcription IRF4 et NFAT-1. Au total, mes travaux de thèse mettent en exergue deux molécules IL-33 et PKC-θ qui pourraient constituer des cibles thérapeutiques potentiellesStudies from our laboratory have shown a critical role of NLRP3 inflammasome in response to ovalbumin allergen. In the present study we investigate the role of NLRP3 and caspase-1 in a mouse model of pulmonary inflammation induced by HDM. We have shown a regulatory role of caspase-1 dependant of the NLRP3 inflammasome and the adaptator molecule ASC but not NLRC4. The regulation of the allergic response is characterized by an increase of eosinophilia, bronchial hyperreactivity and Th2 cytokines production (IL-4, IL-5, IL-13 and IL-33) in lungs. We have shown that mechanisms responsible of this regulation are associated with IL-33 production by macrophages and that neutralization of IL-33 by local administration of a fusion protein of the ST2 receptor (muST2-Fc) reduce characteristics of asthma. These results suggest that caspase-1 activation reduce IL-33 production in vivo regulating lung inflammation and Th2 response induced by HDM. Moreover, we investigate the role of the Protein Kinase C theta (PKC-θ) in allergic airway inflammation. We have demonstrated that PKC-θ plays a protective role in allergic asthma but is critical for the activation and proliferation of innate lymphoid cells (ILC2). In addition, in vivo inhibition by oral administration of PKC-θ specific inhibitor C20 (BIX02656) reduces pulmonary inflammation with IL-5 and IL-13 production. We suggest that PKC-θ is implicated in Th2 and ILC2 differenciation by a mechanism dependant on transcription factors IRF4 and NFAT-1. Finally, my thesis projects describe IL-33 and PKC-θ as potential therapeutic targets for allergic lung inflammation
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Reynders, Ana. "Gut NKp46+ Cells : new members of the emerging family of intestinal lymphoid cells." Thesis, Aix-Marseille 2, 2010. http://www.theses.fr/2010AIX22090/document.
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Nous avons montré que, dans l'intestin, les cellules exprimant le marqueur spécifique des lymphocytes Natural Killer (NK), NKp46 peuvent être divisées en deux compartiments, par rapport au facteur de transcription RORyt. Les cellules NKp46+RORyt- sont des cellules NK intestinales immatures. Cependant, les cellules NKp46+RORyt- s'apparentent aux cellules "lymphoid tissue-inducer" (LTi), puisqu'elles requièrent RORyt pour leur développement et produisent de l'IL-22, cytokine essentielle pour l'homéostasie et la réponse antimicrobienne épithéliale. Notre recherche actuelle vise à établir les relations ontogéniques et la signature génétique de ces cellules au niveau pan-génomique. La fonction in vivo des cellules NKp46+ intestinales est adressée dans un modèle unique d'infection par voie orale avec Listeria monocytogenes. L'ensemble de nos résultats a montré que les cellules NKp46+ ont une place légitime dans la famille émergente des cellules intestinales de l'immunité innéeNatural Killer (NK) cells are NKp46+CD3- innate lymphocytes, which exhibit cytotoxicityand cytokine production, mainly IFN-γ, as major effector functions. In mammals, theexpression of natural cytotoxic receptor NKp46 is essentially restricted to NK cellcompartment. However, we showed that in mouse intestine, NKp46 marker defines aheterogeneous cell population, differentially expressing the retinoic acid orphan receptorRORγt. NKp46+RORγt- cells harbor reduced cytotoxicity and IFN-γ production, consistentwith an immature NK cell phenotype. In contrast, NKp46+RORγt+ cells resemble lymphoidtissue inducer cells (LTi) in their developmental requirement for RORγt and their ability toproduce IL-22. This cytokine is critical for epithelial homeostasis and antimicrobial responseat several epithelial sites.By using a genome wide profiling approach, we confirmed that gut NKp46+RORγt- cellsare the only gut NKp46+ cell population related to conventional splenic NK cells, while gutNKp46+RORγt+ cells are linked to LTi cells. Transcriptional signatures specific of distinct gutNKp46+ cell subsets are currently under intensive investigation in order to determine novelfunctional properties.We assessed gut NKp46+ cell subsets in vivo function during oral infection with entericpathogen Listeria monocytogenes (L.m.). Although immune responses to systemic L.minfection have been widely characterized in mice, L.m. cannot breach mouse intestinal barrier,thus limiting the knowledge of early immune responses at the natural site of infection. Using aunique transgenic mouse lineage restoring L.m. intestinal invasiveness, we showed that gutNKp46+RORγt- and NKp46+RORγ t+ cells respond to L.m. infection by producing IFN-γ andIL-22, respectively. Further investigation of the cellular mechanisms leading to gut NKp46+cell subset activation, as well as the contribution of these cells to the control of bacterialdissemination is in progress.Altogether our data provide novel insight into intestinal innate immunity and highlight gutNKp46+ cell subsets as “legitimate” members of the emerging family of intestinal innatelymphoid cells
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Capobianco, Marcela Petrolini [UNESP]. "Associação de polimorfismos do receptor TCR e dos genes da IL1 e IL2 com a infecção por Plasmodium vivax no município de Goianésia do Pará, Estado do Pará." Universidade Estadual Paulista (UNESP), 2017. http://hdl.handle.net/11449/150430.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
No Brasil o Plasmodium vivax é a espécie mais prevalente, responsável por aproximadamente 85% dos casos de malária. Ademais as variantes da proteína circumesporozoíta (CSP - VK210, VK247 e P. vivax-like) já foram identificadas em várias áreas endêmicas no país. Diversos estudos analisando a influência da variabilidade genética de receptores celulares e moléculas envolvidas na resposta imune, com diferentes peptídeos do Plasmodium, têm obtido resultados variáveis de acordo com o antígeno utilizado e a população analisada. Este trabalho apresenta resultados sobre o estudo de polimorfismos genéticos no TCR (T cell Receptor) e nas Interleucinas 1 e 2 em pacientes infectados por P. vivax provenientes do município de Goianésia do Pará, no Estado do Pará. Avaliou-se estes polimorfismos com a parasitemia do indivíduo, com os genótipos da CSP e com a resposta sorológica contra os peptídeos da CSP. Foram relacionados também estes polimorfismos com os níveis de citocinas. Os polimorfismos foram analisados por técnicas de PCR-RFLP e PCR alelo específico. As análises sorológicas da CSP foram realizadas por ELISA. Foram comparadas as frequências genotípicas observadas segundo o teorema de Hardy-Weinberg (HW). Os níveis de IL1 e IL2 foram avaliados por citometria de fluxo, seguindo protocolo descrito pelo fabricante. Associação dos polimorfismos com os níveis de interleucinas foi avaliada por Análise de variância. As frequências genotípicas e alélicas foram obtidas no programa R v 2.11.1. A parasitemia variou de 15 a 70.000, com mediana de 1.500 parasitos/mm3. Os SNPS investigados mostraram frequências variadas na amostra estudada. Todo o polimorfismo avaliado encontra-se em Equilíbrio de Hard Wenberg. Não houve diferença significativa da parasitemia em relação aos SNPs investigados. Infecções contendo apenas a variante VK247 foram as mais comuns e também não foi observado diferença significativa na resposta de anticorpos de acordo com a variante da CSP presente no momento da infecção. Correlações significativas entre os níveis destas interleucinas com a parasitemia e os níveis de anticorpos contra as variantes da CSP não foram observadas. Além disso, as variantes da CSP não influenciaram os níveis plasmáticos das citocinas e não houve associações positivas entre os SNPs das IL1 e IL2 e seus níveis plasmáticos. Os resultados poderão contribuir na identificação e participação efetiva de genes humanos na modulação da resposta imune, essenciais no estabelecimento de estratégias de imunização contra a doença, em área de transmissão ativa no Estado do Pará.
In Brazil, Plasmodium vivax is the most prevalent species responsible for approximately 85% of malaria cases. In addition, variants of the circosporozoite protein (CSP - VK210, VK247 and P. vivax - like) have already been identified in several endemic areas in the country. Several studies analyzing the influence of the genetic variability of cellular receptors and molecules involved in the immune response with different Plasmodium peptides have obtained variable results according to the antigen used and the analyzed population. This work presents results on the study of genetic polymorphisms in TCR (T cell Receptor) and in Interleukins 1 and 2 in patients infected by P. vivax from the city of Goianésia do Pará, in the State of Pará. These polymorphisms were evaluated with Parasitemia, CSP genotypes and serological response to CSP peptides. These polymorphisms were also related to cytokine levels. Polymorphisms were analyzed by PCR-RFLP and allele-specific PCR techniques. Serological tests of CSP were performed by ELISA. The genotypic frequencies observed according to the Hardy-Weinberg (HW) theorem were compared. Levels of IL1 and IL2 were evaluated by flow cytometry following the protocol described by the manufacturer. Association of polymorphisms with interleukin levels was evaluated by analysis of variance. The genotypic and allelic frequencies were obtained in program R v 2.11.1. The parasitemia ranged from 15 to 70,000, with a median of 1,500 parasites / mm3. The SNPS investigated showed varied frequencies in the sample studied. All polymorphism evaluated is in Hard Wenberg Equilibrium. There was no significant difference in parasitemia in relation to the investigated SNPs. Infections containing only the VK247 variant were the most common and also no significant difference in antibody response was observed according to the CSP variant present at the time of infection. Significant correlations between the levels of these interleukins with parasitemia and levels of antibodies against variants of CSP were not observed. In addition, the CSP variants did not influence plasma cytokine levels and there were no positive associations between IL1 and IL2 SNPs and their plasma levels. The results may contribute to the identification and effective participation of human genes in the modulation of the immune response, essential in the establishment of immunization strategies against the disease, in an active transmission area in the State of Pará.
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Gumà, Uriel Mònica. "Análisis del repertorio de receptores de células NK en la infección por citomegalovirus humano." Doctoral thesis, Universitat Pompeu Fabra, 2005. http://hdl.handle.net/10803/7092.
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Los objetivos de este trabajo han sido estudiar la expresión de receptores de células NK (NKR), en particular CD94/NKG2C, en relación con la infección por citomegalovirus humano (HCMV). Los resultados descritos constituyen la primera evidencia de que la infección por HCMV modifica el repertorio de NKR. El incremento en la proporción de células CD94/NKG2C+ en donantes seropositivos para HCMV sugiere que participan en la respuesta al patógeno. El receptor CD94/NKG2C no sólo estimula las funciones efectoras y la proliferación de las células NK, sino que también activa a una subpoblación minoritaria de células T CD8+. El estudio de la expansión in vitro de la subpoblación NK CD94/NKG2C+ tras la interacción con fibroblastos infectados por el HCMV, sugiere que el propio receptor está implicado en la proliferación.The main goals of this work have been to study the influence of human cytomegalovirus (HCMV) infection on the NK cell receptor (NKR) repertoire, mainly on the CD94/NKG2C receptor. Our observations provide a first evidence indicating that human cytomegalovirus (HCMV) may selectively shape the NKR repertoire. The increased proportions of NKG2C+ cells in HCMV-seropositive donors suggest a role in the response against the virus. The CD94/NKG2C receptor triggers the effector functions and proliferation not only in NK cells but also in a subset of CD8+ T lymphocytes. The stimulation of PBL from HCMV+ donors with virus-infected fibroblasts elicited a preferential expansion of CD94/NKG2C+ NK cells; studies carried out in this experimental system suggest that the receptor is involved in driving the proliferation.
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Ahmed, S. A. Abu Tayeh. "Constitutive RIG-I activation causes skin lesion resembling psoriasis in transgenic mice." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263796.
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Link, Lisa [Verfasser], and Ernst Rainer [Akademischer Betreuer] Weissenbacher. "Verhalten der Interleukine IL4, IL8 und IL12 aus dem Vaginalsekret von Patientinnen mit symptomatischer Vaginitis / Lisa Link ; Betreuer: Ernst Rainer Weissenbacher." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1121507905/34.
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Durost, Philip A. "Evaluation of IL2 and HLA on the Homeostasis and Function of Human CD4 and CD8 T Cells." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/936.
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Homeostasis of human T cells is regulated by many factors that control proliferation, differentiation of effector cells and generation of memory. Our current knowledge of the mechanisms controlling human T cell homeostasis in vivo is based on experiments in small animal models. However many differences exist between immune systems of mice and humans, including cell composition, function, and gene expression. Humanized mouse models have shown great value in the study of human immunobiology. I have used novel humanized mouse models to examine the role of human MHC (HLA) and human IL2 in CD8 T cell and CD4 regulatory T cell (Treg) homeostasis. To study human CD8 T cells I engrafted CD8 T cells from healthy donor PBMC into NOD-scid IL2rgnull (NSG) mice that lacked expression of murine MHC and that expressed HLA-A2. My data demonstrate that CD8 T cell survival and effector function required the presence of HLA-A2, helper function from human CD4 T cells and exogenous human IL2. To study human Treg homeostasis I used NSG mice engrafted with human fetal thymus and hematopoietic stem cells (BLT model). NSG-BLT mice support the growth of human thymic tissue and enable the efficient development of HLA-restricted Treg and conventional T cells. Using an AAV vector to express human IL2, I demonstrated that functional human Treg but not conventional T cells increased in number in NSG-BLT mice and that this coincided with increases in activated human NK cells. Overall my research has revealed that HLA and human IL2 have an essential role in human T cell survival and function in vivo.
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Nataf, Éric. "Etude cytogenetique du myelome multiple : a propos de 49 patients ; interet de l'utilisation gm. - csf - il6 - il3." Lille 2, 1992. http://www.theses.fr/1992LIL2M180.
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Durost, Philip A. "Evaluation of IL2 and HLA on the Homeostasis and Function of Human CD4 and CD8 T Cells." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/936.
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Homeostasis of human T cells is regulated by many factors that control proliferation, differentiation of effector cells and generation of memory. Our current knowledge of the mechanisms controlling human T cell homeostasis in vivo is based on experiments in small animal models. However many differences exist between immune systems of mice and humans, including cell composition, function, and gene expression. Humanized mouse models have shown great value in the study of human immunobiology. I have used novel humanized mouse models to examine the role of human MHC (HLA) and human IL2 in CD8 T cell and CD4 regulatory T cell (Treg) homeostasis. To study human CD8 T cells I engrafted CD8 T cells from healthy donor PBMC into NOD-scid IL2rgnull (NSG) mice that lacked expression of murine MHC and that expressed HLA-A2. My data demonstrate that CD8 T cell survival and effector function required the presence of HLA-A2, helper function from human CD4 T cells and exogenous human IL2. To study human Treg homeostasis I used NSG mice engrafted with human fetal thymus and hematopoietic stem cells (BLT model). NSG-BLT mice support the growth of human thymic tissue and enable the efficient development of HLA-restricted Treg and conventional T cells. Using an AAV vector to express human IL2, I demonstrated that functional human Treg but not conventional T cells increased in number in NSG-BLT mice and that this coincided with increases in activated human NK cells. Overall my research has revealed that HLA and human IL2 have an essential role in human T cell survival and function in vivo.
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Silva, Helker Albuquerque Macedo da. "Polimorfismos de base única em genes de interleucinas no lúpus eritematoso." Universidade Federal de Pernambuco, 2014. https://repositorio.ufpe.br/handle/123456789/12522.
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O Lúpus eritematoso sistêmico (LES) é uma doença autoimune sistêmica e crônica, com uma patogênese envolvendo múltiplos fatores. As citocinas têm um papel crucial no desenvolvimento e progressão do LES. O objetivo do presente trabalho foi avaliar a associação dos polimorfismos dos genes das interleucinas proinflamatórias IL2, IL12B, IL17A, IL17F, IL23R e TNF com o desenvolvimento do LES, atividade da doença e manifestações clínicas apresentadas. Foram selecionadas 122 mulheres com Lupus atendidas no Hospital das Clínicas-UFPE. Os polimorfismos TNF (-308 G/A), IL17A -197(G/A), IL17F (7488 A/G), IL23R (2199 A/C) e IL12B (3’UTR +1188 A/C) foram identificados por PCR-RFLP e a genotipagem do polimorfismo 3’UTR +1188 (A/C) IL2 foi por ARMS-PCR. Nossos resultados mostram que os polimorfismos dos genes TNF (p=0,0012), IL17F (p=0,0005), IL2 (p=0,0011), IL12 (p=<0,0001) e IL23R (p=<0,0001) estão associados à suscetibilidade ao LES. Nenhum dos polimorfismos mostrou associação com o nível de atividade da doença. Na análise de associação entre os polimorfismos e o desenvolvimento de características clínicas, o alelo A do TNF mostrou associação com o desenvolvimento de Serosite (p=0,0228). Além disso, observou-se que polimorfismo do gene IL12B mostrou associação com Fator Anti-nuclear (FAN) (p=<0,0001). Desta forma, concluímos que, na população estudada, polimorfismos em genes de citocinas próinflamatórias e seus receptores podem ser fatores de risco para o desenvolvimento do LES, mas não se associam com a atividade da doença, e que os polimorfismos nos genes TNF e IL12B podem influenciar no desenvolvimento de características clínicas da doença.
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Hacein-Bey-Abina, Salima. "Obtention d'un anticorps monoclonal dirigé contre la chaine g du récepteur à l'IL2 : tentative et mise au point." Paris 5, 1993. http://www.theses.fr/1993PA05P192.
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Baudhuin, Jérémy. "Régulation de l'activité d'effecteurs cellulaires de la réponse immunitaire innée par interaction des récepteurs inhibiteurs ILT2 et ILT4 avec la molécule HLA-G." Paris 7, 2013. http://www.theses.fr/2013PA077207.
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La subversion ou le dysfonctionnement des processus de tolérance de l'immunité innée peuvent conduire au développement de diverses pathologies. D'une part, en interagissant avec le récepteur inhibiteur ILT2 exprimé à la surface des cellules NK et des lymphocytes T y8, la molécule HLA-G permet aux cellules tumorales l'exprimant d'échapper à l'activité anti-tumorale de ces deux populations. En effet, en perturbant l'organisation de la synapse immunologique, et ce indépendamment des radeaux lipidiques, HLA-G entraîne l'inhibition de l'activité cytotoxique des cellules NK. De plus, elle provoque l'inhibition de la prolifération, de la synthèse d'IFN-y et de la cytotoxicité des lymphocytes T y& D'autre part, l'engagement du récepteur ILT4, autre récepteur connu de HLA-G, à la surface des granulocytes neutrophiles, conduit à une diminution de leur fonction phagocytaire et oxydative induites par le récepteur CD32a. Contenu dans les granules intracytoplasmiques des neutrophiles, ILT4 peut être mobilisé en surface par exocytose en réponse à un stimulus pro-inflammatoire, contribuant ainsi à la régulation de leur activité inflammatoire. L'utilisation d'échantillons de patients atteints de sepsis indique que ce mécanisme de surexpression de ILT4 est perturbé dans un contexte inflammatoire et suggère un impact sur l'activité des neutrophiles au cours de ce syndrome. L'ensemble de ces études nous permet d'envisager les récepteurs ILT2 et ILT4 ainsi que leur ligand HLA-G comme des cibles thérapeutiques dans les traitements de patients atteints de cancers ou comme outils thérapeutiques dans le traitement de pathologies inflammatoiresDisruption or subversion of innate immunity tolerogenic mechanisms can lead to several diseases. On one hand, interaction of ILT2 inhibitory receptor expressed on NK and y8 T cells with HLA-G molecule expressed on tumor cells enable these last to escape from the antitumor activity of these two populations. Indeed, independently from tumor lipid rafts integrity, HLA-G prevents the organisation of NK cells immunological synapse and therefore inhibits their cytolytic activity. Moreover, it induces the inhibition of y5 T cells proliferation, IFN-y synthesis and cytotoxicity. On the other hand, engagement of ILT4, another inhibitory receptor for HLA-G, on neutrophils surface, entails the decrease of both CD32a-dependent phagocytic and oxidative functions. ILT4 is also contained in intracytoplasmic granules and its surface expression can consequently be increased through exocytosis following a pro-inflammatory stimulus. By this way, exocytosis contributes to the regulation of neutrophil activity. The use of sepsis patients' samples indicates that ILT4 up-regulation is disrupted in an inflammatory context and suggests an impact on neutrophils activity in this syndrome. Overall, these studies enable us to propose HLA-G and its receptors ILT2 and ILT4 as new therapeutic targets to optimize immunotherapy treatments for cancer patients or as therapeutic tools in the treatment of inflammatory disorders
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Cunha, Wilton Darleans dos Santos. "Influência do exercício sobre a resposta imunológica de ratos desnutridos." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-18112009-115338/.
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A desnutrição é capaz de induzir diversas alterações metabólicas afetando marcadamente a composição corporal e o sistema imunológico. O exercício físico, por sua vez, produz alterações no organismo para uma melhor capacidade de adaptação a situações de estresse. O desvio da situação de homeostase produzida pelo exercício físico induz uma reorganização de seus mecanismos funcionais, principalmente dos mecanismos endócrinos e imunológicos. Ainda é pouco conhecida a influência do exercício sobre a desnutrição e também as conseqüências sobre o sistema imunológico quando as duas variáveis são combinadas. Assim, esse trabalho teve como objetivo avaliar os efeitos do exercício físico de endurance sobre ratos submetidos a um protocolo de desnutrição crônica. Avaliamos ratos Wistar machos, desnutridos por 16 semanas, divididos em 4 grupos: eutrófico sedentário (ES), eutrófico treinado (ET), desnutrido sedentário (DS), desnutrido treinado (DT). O treinamento físico foi realizado em esteira, por 10 semanas, 5 vezes por semana, com intensidade aproximada de 60- 65% do consumo máximo de oxigênio. Avaliou-se a composição corporal, através da aferição do peso corporal, peso dos tecidos muscular esquelético e adiposo, do fígado, do conteúdo de gordura e proteína na carcaça, e a concentração de leptina, ACTH, glicose, insulina, e glutamina no plasma. Avaliamos também, através de citometria de fluxo, os marcadores de superfície celular CD3 e CD4, bem como a celularidade no timo. O consumo máximo de oxigênio e o desempenho através de um teste até a exaustão também foram analisados. A análise estatística utilizada foi o teste de variância ANOVA two-way com pós teste de Bonferroni e, nível de significância adotado de p<0,05. Os resultados encontrados demonstraram que o treinamento de endurance em ratos submetidos à desnutrição crônica promoveu uma acentuada redução do peso e da adiposidade corporal; um aumento da massa muscular relativa ao peso corporal; um restabelecimento da glicemia aos valores normais; uma melhor relação da concentração insulina/glicose, sugerindo uma sensibilidade à insulina aumentada; um aumento dos estoques de glicogênio muscular; um maior consumo máximo de oxigênio; e uma recuperação na morfologia e fisiologia tímica, uma maior resposta proliferativa do baço e linfonodos estimulados com IL2. Concluímos desta forma que o exercício foi capaz de recuperar a morfologia, como também a maturação timócitos CD3 e CD4 e sua celuraridade em ratos desnutridos. A resposta proliferativa à estimulação da IL2 também foi recuperada.Malnutrition is capable of inducing diverse metabolic alterations, markedly affecting body composition and the immune system. Physical exercise, on the other hand, induces a renders the organism more capable of adaptation to stress. Still, little is known about the influence of exercise training upon malnutrition-related alterations and its consequences on the immune system. Our aim was to evaluate the effect of moderate intensity exercise training in rats submitted to a protocol (16wk) of chronic malnutrition. Male Wistar rats were divided in to 4 groups: sedentary, fed ad libitum (SF); trained fed ad libitum (TF); sedentary energy restricted (RES); and trained energy restricted (TER). Training was carried out on a treadmill for 10 weeks, 5 time wk, under an intensity of 60-65% of the maximal oxygen consumption. We evaluated the Corporal composition, the variation of body weight, and the weight of the skeletal muscle, adipose tissues, and liver; as well as fat and protein content in the carcass; and also plasma leptin, ACTH, glucose, insulin and glutamine concentration. We also examined through flow cytometry CD3 and CD4, as well as the celularity in the thymus. The maximum consumption of oxygen and the performance were also assessed. The results demonstrate that endurance training in rats submitted to the chronic malnutrition protocol promoted reduction of body weight and of corporal adiposity; an increase in the relative contribution of muscle to body weight; the reestablishment of glicemia; improval of insulin/glucose reason, suggesting increased sensitivy to insulin; an increase of muscle glycogen content; enhanced oxygen consumption; are recovery of the morphology and physiology of the thymus, together with a proliferative response of the spleen and lymph nodes stimulated with IL2. We conclude in such a way that moderate intensity training restored thymus morphology and the capacity of maturation of CD3 and CD4 and also timocyte number and the of proliferative response to IL2 stimulation.
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Demmer, Wolfram [Verfasser]. "Induktion einer tumorspezifischen Immunantwort zur Therapie des HCC durch CD40L- und IL2-exprimierende Dendritische Zellen, in vivo / Wolfram Demmer." Bonn : Universitäts- und Landesbibliothek Bonn, 2012. http://d-nb.info/1043510230/34.
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Beuraud, Chloé. "Identification et caractérisation d'une population de cellules lymphoïdes innées de type 2 (ILC2) associée à la sévérité de la rhinite allergique et de l'asthme." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS475.
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Identification et caractérisation d'une population d'ILC2 associée à la sévérité de la rhinite allergique et de l'asthmeTrois catégories de cellules lymphoïdes innées (innate lymphoid cells, ILC) ont été décrites récemment sur la base de leurs phénotypes et leurs caractéristiques fonctionnelles : les ILC1, ILC2 et ILC3. Les ILC2 semblent avoir un rôle pro-inflammatoire important dans l’allergie en raison de leur capacité à produire de grandes quantités de cytokines TH2.Pour mieux comprendre le rôle de ces cellules dans l’allergie respiratoire, nous avons comparé les ILC sanguines de patients atteints d’une rhinite allergique associée ou non à un asthme, à celles de sujets non allergiques. Cette étude révèle de multiples différences fonctionnelles entre les ILC circulantes de sujets sains et allergiques. Notamment, la fréquence d’ILC2 exprimant le récepteur aux chimiokines CCR10 est augmentée dans le sang de patients asthmatiques sévères.CCR10 pouvant permettre le recrutement des ILC vers les organes cibles, le rôle des ILC2 CCR10+ dans la physiopathologie de l’asthme a été étudié. Leur présence dans les poumons humains a été observée. Des analyses fonctionnelles et phénotypiques ont révélé que cette sous-population cellulaire était peu activée mais présentait une plasticité leur conférant des caractéristiques proches des ILC1. La déplétion de ces cellules dans un modèle murin d’asthme allergique aggrave l’hyperréactivité bronchique.Les travaux de cette thèse documentent le rôle des ILC dans l’asthme. En particulier, la fréquence sanguine d’ILC2 CCR10+ augmente avec la sévérité de la maladie. Les résultats obtenus dans les modèles animaux suggèrent que ces cellules auraient un rôle bénéfique dans le contrôle de l’asthme. La voie du CCR10 pourrait représenter une nouvelle cible pour le développement de traitements innovants contre l’asthme ou une source prometteuse de biomarqueursIdentification and characterization of an ILC2 subset linked to allergic rhinitis and asthma severityInnate lymphoid cells (ILCs) have been classified into ILC1, ILC2 and ILC3 subsets based on their respective phenotypes and functions. Considering the strong ability of ILC2s to produce TH2 cytokines, these cells likely play a significant role in allergic diseases.To better understand the role of these cells in respiratory allergies, we compared blood ILCs from allergic patients with or without asthma to non-allergic individuals. Together our results show multiple functional differences between ILC from allergic and healthy subjects. In particular, ILC2s expressing the chemokine receptor CCR10 are specifically enriched in the blood of patients with severe allergic asthma.Considering that CCR10 could allow the recruitment of ILCs to target organs, the role of CCR10+ ILC2s in asthma physiopathology has been studied. This ILC2 subtype is present in human lungs. Functional and phenotypic analyses revealed that these cells are less activated than other ILC2s and show ILC1-like properties. CCR10+ ILC2s depletion in a mouse model of allergic asthma exacerbate airway hyperreactivity.Together, this work documents the role of ILCs in asthma. Specifically, circulating CCR10+ ILC2 frequency increases with asthma severity. The results obtained in mouse models suggest that these cells could have a beneficial role in asthma control. CCR10 pathway could represent a new target to elaborate breakthrough treatments against asthma or a source of promising biomarkers
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Alilèche, Abdelkrim. "Étude de l'expression et de la fonction du système IL2/IL2R par les cellules fibroblastiques et les cellules de mélanome." Paris 11, 1994. http://www.theses.fr/1994PA114801.
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Espígol, Friigolé Georgina. "Diferenciació funcional Th17 en l'expressió fenotípica i evolució clínica de l'arteritis de cèl•lules gegants. Rol de l'axis IL23/IL-17 en la inflamació vascular." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/123212.
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L’arteritis de cèl.lules gegants (ACG) és una vasculitis granulomatosa que afecta a artèries de mitjà i gran calibre amb una predilecció especial per les branques de la caròtida.
La malaltia acostuma a respondre inicialment de manera satisfactòria al tractament amb glucocorticoids (GC) tot i que sovint els pacients requereixen un tractament prolongat i a vegades indefinit amb la iatrogènia que aquest fet comporta.
Existeixen evidències de que la major part de manifestacions clíniques i complicacions isquèmiques dels pacients són atribuibles als potents efectes biològics que les citocines i factors de creixement exerceixen sobre la paret vascular i també a distància originant una resposta inflamatòria sistèmica. A nivell dels petits vasos que envolten l’adventicia de l’artèria, es produeixen interaccions complexes entre l´endoteli i els leucòcits amb activació de les cèl.lules inflamatòries Els limfòcits i els macròfags activats produeixen citocines proinflamatòries (TNFα i IL-1) que entre d’altres accions, indueixen l’expressió de molècules d’adhesió de l’endoteli vascular. Els factors de creixement afavoreixen l’angiogènesi i els vasos neoformats incrementen la superfície d’interacció amb les cèl•lules inflamatòries.
S’ha demostrat que les interleucines IL-12, IL-23 i IL-17 tenen un rol molt destacat en la inflamació crònica i són també dianes terapèutiques en algunes malalties, l’objectiu principal d’aquesta tesi és estudiar el seu paper en l’ACG.
Objectius i Mètodes:
1. Investigar l´expressió de p40/p35 (IL-12) i p19/p40 (IL-23) i IL-17 en artèries temporals de pacients amb arteritis de cèl.lules gegants, tractats i seguits prospectivament mitjançant tècniques de RT- PCR quantitativa a temps real i immunohistoquímica.
2. Determinar les concentracions de p40, p35, p19 i IL-17 en sèrum de pacients amb ACG obtinguts abans d’iniciar el tractament mitjançant tècniques d´ELISA.
3. Estudiar la possible correlació entre l’expressió arterial i concentracions sèriques d´aquestes citocines, la intensitat de la resposta inflamatòria sistèmica en el moment del diagnòstic i la resposta al tractament en una sèrie àmplia de pacients tractats i seguits prospectivament.
4. Investigar mitjançant RT-PCR quantitativa, ELISA i western-blot, la producció de les citocines IL12-IL23 en cèl•lules endotelials estimulades amb lligants dels TLRs com LPS entre d´altres.
5. Induir sobreexpressió de IL23p19 en les cèl•lules endotelials i estudi dels seus efectes en la funció inflamatòria de les mateixes, estudiant canvis en el mRNA de gens inflamatoris d’aquestes cèl•lules mitjançant PCR quantitativa.
6. Estudiar l´efecte de la sobreexpressió de IL23p19 en les cèl.lules endotelials i els seus efectes en respostes relacionades amb l´angiogènesi com a mecanisme de persistència de la inflamació, mitjançant estudis in vitro de migració i diferenciació.
7. Determinar l´efecte in vivo de la IL23p19 en les cèl.lules endotelials mitjançant l´implant de plaques de matrigel amb injecció de cèl.lules endotelials amb sobreexpressió de p19 i estudiar diferències en angiogènesi mitjançant immunotinció.
Resultats:
1. Les interleucines IL-12 i IL-23 es troben altament expressades en l’ACG tan en forma de mRNA com de proteina a nivell local. No hem pogut establir la seva relació amb les característiques fenotípiques dels pacients i pel que fa a la persistència de la malaltia nivells elevats inicials en el mRNA de la subunitat p40, semblen relacionar-se amb una millor resposta al tractament.
2. Correlació entre els nivells de mRNA d’IL-23 i els d’IL-17 en l’ACG i una important expressió d’IL-17 en aquesta malaltia. Contràriament al que podíem esperar, hem detectat la IL-17 com a factor predictor de bon pronòstic, pacients amb nivells més alts de IL-17mRNA abandonen abans el tractament de forma significativa.
3. Estimuls inflamatoris com el TNFα o l’IFNγ afavoreixen la producció d’IL-23p19 per part de les cèl.lules endotelials. No hem pogut detectar la co-producció de p40, en principi necessària per la secreció de la citocina amb activitat biològica (IL-23).
4. La IL-23p19 derivada de les cèl.lules endotelials pot ser funcional, tot i que amb els nostres resultats encara no podem establir cap mecanisme que ho expliqui.Background: The giant cell arteritis (GCA) is a granulomatous vasculitis affecting large and medium sized vessels with a special predilection for branches of the carotid artery. Most patients respond dramatically to high-dose corticosteroids, but the subsequent outcome is highly variable. Corticosteroid-related side effects are frequent during the follow-up of GCA patients. Although corticosteroids induce an important symptomatic improvement, they appear to be insufficient to completely suppress essential pathways involved in disease persistence. IL-12, IL-23 and IL-17 play an important role in chronic inflammation and they are therapeutic targets in some autoimmune diseases. Our aim was to study the role of these cytokines in GCA. Objectives and Methods: 1. To investigate IL-12 p40, IL-12 p35, IL-23 p19 and IL-17A expression in temporal artery lesions from patients with giant-cell arteritis (GCA), and its relationship with disease outcome. (RT-PCR, ELISA, Immunostaining) 2. To investigate by real-time quantitative PCR, immunostaining, ELISA and western-blot production of IL12 and IL23 cytokines in endothelial cells following stimulation with TLRs ligands as LPS (TLR4) and others. 3. To study the effect of IL23 p19 overexpression in pro-inflammatory and angiogenic functions of endotelial cells (EC). By real-time quantitative RT-PCR of inflammatory genes and in vitro angiogenesis assays (migration and differentiation). 4. To determine in vivo the effect of overexpression of IL23 p19 in endotelial cells by injection of matrigel plugs with EC and to study differences in angiogenesis by immunostaining. Results: 1. IL-12 and IL-23 were significantly more abundant in temporal arteries from untreated patients than in arteries from controls. We found a lack of correlation between the expression of these cytokines and systemic inflammatory findings. 2. IL-17A expression was significantly increased in temporal artery samples from GCA patients compared to controls. Surprisingly, patients with strong IL-17A expression tended to experience less relapses, and required significantly shorter treatment periods. 3. Estimuls inflamatoris com el TNFα o l’IFNγ afavoreixen la producció d’IL-23p19 per part de les cèl.lules endotelials. No hem pogut detectar la co-producció de p40, en principi necessària per la secreció de la citocina amb activitat biològica (IL-23). 4. La IL-23p19 derivada de les cèl.lules endotelials pot ser funcional, tot i que amb els nostres resultats encara no podem establir cap mecanisme que ho expliqui
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Miralles, Consuegra Marta. "Inmunomodulació de la vía Th17 con vectores virales portadores del receptor soluble de IL23: una nueva estrategia de terapia génica para el tratamiento de sclerosis múltiple." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/283733.
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En las últimas décadas, la incidencia de enfermedades autoinmunes ha aumentado notablemente, ya sea por el mayor conocimiento, por la mejora en las herramientas de diagnóstico, o por los cambios en los hábitos de vida. Así, el incremento de afectados por este tipo de enfermedades se ha convertido en un problema social grave, pues trastornos autoinmunes crónicos como la Esclerosis Múltiple (EM) o la Enfermedad de Crohn (EC) pueden aparecer en etapas tempranas de la vida, afectando gravemente a su calidad. Más aún, los tratamientos actuales contra estas enfermedades son paliativos y frenan levemente su desarrollo, sin lograr evitar su transcurso a largo plazo, y provocando efectos secundarios graves muchas veces. Por otro lado, el coste de los medicamentos y los cuidados multidisciplinares que los pacientes requieren a lo largo de su vida constituyen un problema económico-sanitario elevado, haciendo cada vez más importante la aparición de una cura eficaz.
La EC es una enfermedad inflamatoria intestinal grave que afecta especialmente al colon y al íleo, además de tener otras complicaciones sistémicas y extraintestinales. Por su parte, la EM es una enfermedad desmielinizante que afecta al SNC provocando una pérdida progresiva de la movilidad y otras complicaciones secundarias. En ambas enfermedades juega un papel muy importante la vía Th17, y por consiguiente, la IL23. De hecho, algunos tratamientos experimentales para ambas tratan de inhibir la función de IL23 mediante el uso de anticuerpos, con resultados controvertidos. Así pues, en este trabajo nos propusimos estudiar una estrategia de terapia génica no basada en el uso de anticuerpos, sino en un vector viral portador de IL23R soluble (IL23Rs), que se uniera a IL23 y redujera su acción.
En la primera parte, nos centramos en la EC y en la obtención del vector viral idóneo para terapia intestinal. El adenovirus quimera Ad5/40S, con un marcado tropismo intestinal cumple estos requisitos. Sin embargo, su producción in vitro era muy ineficiente, por lo que se desarrolló un método optimizado de producción, basado en el uso de células 211B en suspensión y polybrene durante las infecciones en el proceso de amplificación viral.
Por otra parte, también se realizaron dos estudios de tropismo con diferentes vectores virales: in vitro en linfocitos T CD4+, demostrando que el Ad5/40S es el vector que mejor infecta, e in vivo, estudiando el tropismo celular en intestino y demostrando que ninguno de los vectores virales infecta eficientemente las stem cells de las criptas de Lieberkühn.
Debido a que no obtuvimos un perfil Th17 en el modelo de colitis inducida por DSS, en la segunda parte del trabajo nos centramos en la EM. Primeramente, se diseñó el IL23Rs murino, que fue clonado en diferentes vectores virales. Seguidamente, se demostró que células infectadas con estos vectores secretaban IL23Rs al exterior celular y que IL23Rs se unía a IL23 in vitro.
Finalmente, los vectores portadores de IL23Rs se probaron en un modelo murino de EM, la Encefalomielitis Autoinmune Experimental. Los resultados expuestos en este trabajo indican que la expresión de IL23Rs tiene un efecto beneficioso en la evolución clínica de la enfermedad, retrasando la aparición de los primeros síntomas y reduciendo la gravedad de los mismos hasta el final del seguimiento clínico. Asimismo, en concordancia con la mejora clínica observada, los ratones tratados no presentan evidencias de desmielinización, además de tener una menor inflamación y una respuesta astroglial y microglial significativamente menor en médula espinal.
En resumen, nuestros resultados sugieren que el empleo de una terapia génica inmunomoduladora para EM basada en el uso de vectores virales portadores de IL23Rs podría tener un efecto terapéutico en el desarrollo de la enfermedad.In the last decades, the incidence of autoimmune diseases has increased significantly, either by the greater knowledge we have of them, by the improvement of diagnostic tools, or by changes in lifestyle. Indeed, the increase of patients affected by these diseases has become a serious social problem since chronic autoimmune disorders like Multiple Sclerosis (MS) or Crohn's Disease (CD) appear early in the lives of patients, affecting seriously their quality of life. Moreover, current treatments for these diseases fail to prevent therapeutic effect at long term and cause serious side effects in many cases; despite they slightly alleviate the symptoms and delay the development of the disease. In addition, the cost of drugs and multidisciplinary care that patients require throughout their life are high economic-health problems. Thus, the development of an effective cure for these diseases has become very important. CD is a severe inflammatory bowel disease affecting colon and ileum, but also other systemic and non-intestinal complications. On the other hand, MS is a demyelinating disease of the central nervous system that causes a progressive loss of mobility and many other systemic symptoms and secondary complications. In both autoimmune diseases the Th17 pathway plays an important role, and therefore the IL23 as well. In fact, the rationale of some experimental treatments for both diseases is based on inhibiting the function of IL23 using antibodies, but unfortunately the reported results are controversial. Thus, in this work we have studied a gene therapy strategy not based on the use of antibodies, but on a viral vector carrying a soluble form of IL23R, which binds the IL23 in order to reduce its action. In the first part of this work we focused on CD and in the generation of an appropriate viral vector for intestinal therapy. The chimeric adenovirus Ad5/40S with marked intestinal tropism is a good candidate vector for this purpose. However, the in vitro production of the Ad5/40S was very inefficient, so an optimized production method was developed. The new method was based on the use of suspension cells 211BS and polybrene during infections in the viral amplification process. Moreover, a tropism study was conducted with CD4+ T lymphocytes, demonstrating that the Ad5/40S is the vector infecting these cells with the highest efficiency. A study of the intestinal cell tropism in vivo of different viral vectors was also performed, showing that none of them infects stem cells in the crypts of Lieberkühn. In the second part of this work we focused on MS. First, we designed the soluble form of murine IL23R (IL23Rs), which was cloned in different viral vectors. Subsequently, it was demonstrated that cells infected with these viruses secreted IL23Rs to the extracellular medium. It was also demonstrated that IL23Rs is able to bind to IL23 in vitro. Finally, vectors carrying IL23Rs were tested in the mouse model of MS, Experimental Autoimmune Encephalomyelitis. After several approaches, we demonstrated that expression of IL23Rs has a positive effect on the clinical course of the disease, delaying the time of onset and reducing the severity of symptoms until the end of the experiment. In agreement with the clinical improvement observed, treated mice have a reduced inflammatory response, a significantly lower astroglial and microglial activation as well as an absence of demyelinating signs in spinal cord. In summary, our results suggest that the use of an immunomodulatory gene therapy for multiple sclerosis based on the use of viral vectors carrying IL23Rs could have a therapeutic effect in the development of the disease.
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Hartmann, Katja [Verfasser]. "Genetische Diversität bei europäischen, asiatischen und afrikanischen Schaf- und Ziegenrassen am Kappa-Kasein-(CSN3) und Interleukin-2-Genort (IL2) / Katja Hartmann." Gießen : Universitätsbibliothek, 2011. http://d-nb.info/1061195511/34.
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Gironda, Martínez Adrián. "DNA-ENCODED CHEMICAL LIBRARIES: ADVANCES AND APPLICATIONS TO DRUG DIFFICULT TARGETS." Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1194175.
Full textAbstract:
The discovery of small organic ligands or biologics capable of modulating biological processes remains one of the biggest challenges in developing new medicines. Different technologies have been implemented over the last decades to ease this process and make it more efficient. In this regard, encoded display technologies have played a major role in the discovery of new antibodies, peptides, and proteins. However, the efficient exploitation of automated high-throughput screening to discover small organic ligands has mainly been limited to big pharmaceutical companies. DNA-Encoded Chemical Libraries (DELs) have emerged as a powerful and cost-effective alternative to solve this issue. The technology has been established during the last 25 years and has become one of the best methods to synthesize and screen libraries of unprecedented size, promising a bright future in the early drug discovery stages.
DELs are collections of small molecules individually coupled to oligonucleotide fragments, serving as amplifiable identification barcodes. In the first part of this thesis new DEL designs, displaying molecules capable of targeting challenging therapeutic targets while keeping library-quality at the highest grade, were investigated. A novel single-pharmacophore library, termed AG-DEL, was synthesized. The library was constructed using split-and-pool procedures on single-stranded DNA. The modularity of this library design allowed the creation of different dual-pharmacophore libraries in an encoded self-assembling chemical library format (ESAC 2+1 and ESAC Plus). Furthermore, the new AG-DEL facilitated the use of novel screening methodologies (e.g., photo-crosslinking) to efficiently discover new small organic ligands.
DEL synthesis mainly relies on the chemical diversity of building blocks and the efficiency of the chemical reactions to link them. Following this trend, many different groups have made great efforts during the last years to develop new mild and efficient DNA-compatible reactions. One of the most used reactions for DEL synthesis is the amide bond formation, thanks mainly to various reliable reaction protocols and the big commercially available collections of amino acids. Nevertheless, the current availability of DNA-compatible post-functionalization of amino acids is still quite limited due to some restrictions inherent to the presence of the DNA. In the second part of this thesis, a new DNA-compatible diazo-transfer reaction was successfully optimized and implemented. This reaction has shown to be efficient, both in reaction times and reaction yields, as well as to be mild and fully compatible with DNA, as demonstrated by subsequent enzyme-mediated ligation of the oligonucleotide template to a new fragment, and has served for the synthesis of new ESAC Plus libraries within our group.
The modulation of protein-protein interactions (PPIs) represents another formidable challenge. These interactions are often characterized by large and flat protein surfaces that are composed of many different interacting groups. Therefore, these interactions are usually targeted using large macrocyclic peptides or antibodies. Notwithstanding this challenge, some examples have been reported during the last years in which small organic ligands or peptidomimetics were specifically designed for targeting this class of proteins. Some of these examples have successfully reached clinical trials and even marketing authorization, showing the critical importance of PPI modulators and indicating broad prospects.
In PPI modulation, the discovery of ligands targeting cytokines is even more challenging, due to the small size and particularly flat surface of these proteins. Nevertheless, different small molecule ligands targeting cytokines have been described over the years. Among all these proteins, Interleukin-2 (IL2) represents one of the best examples. IL2 is a pro-inflammatory cytokine, that plays a crucial role in immunity, and different therapeutic approaches using IL2 are increasingly being used for the treatment of a variety of malignancies, like melanoma and renal cell carcinoma. However, the use of IL2 has been limited due to strong side effects related to the high doses of cytokine necessary to achieve a pharmacological effect. Side effects have been linked to the release of pro-inflammatory cytokines as well as to CD25-mediated endothelial damage induced by IL2 binding to endothelial surface receptors, leading to a vascular leak syndrome. The interaction between IL2 and its alpha subunit receptor (IL2Ra or CD25) activates immunosuppressive regulatory T cells (Tregs) and reduces its antitumor activity. Thus, avoiding the formation of the multimeric IL2/IL2R complex can enhance the antitumor response.
The last chapter of this thesis was focused on the development of novel DEL-derived IL2 ligands capable of interacting at the CD25 binding domain of IL2. During these studies, a tumor-targeting antibody-IL2 fusion protein, L19-IL2, was used to find ligands masking the IL2 moiety. The ligands were optimized by a medicinal chemistry approach and characterized by fluorescence polarization. Furthermore, the best ligand showed binding at the CD25 binding epitope of IL2, as evidenced by competition experiments using an anti-IL2 antibody. The use of one of the discovered compounds or an affinity matured derivative can allow the generation of a new class of biopharmaceutical-small molecule complexes that localize at the site of the disease and regain activity of the cytokine only at the tumor site.