Orientador: Carlos Rossa Junior / Banca: Luis Carlos Spolidório / Banca: Silvana Regina Perez Orrico / Banca: Dagmar Ruth Stach Machado / Banca: Paula Cristina Trevilatto / Resumo: Embora a doença periodontal tenha origem infecciosa, ela se caracteriza por uma complexa resposta imune-inflamatória, com a participação de células residentes e não-residentes, produzindo diversas citocinas e mediadores biológicos. A principal característica da doença periodontal destrutiva é a reabsorção do osso alveolar, a qual é uma consequência frequentemente irreversível do processo patológico e das citocinas produzidas pela resposta do hospedeiro. Citocinas específicas atuam diretamente no controle da remodelação óssea, denominadas RANKL (Receptor activator of NF-kB ligand) e OPG (Osteoprotegerin,). RANKL é uma proteína produzida por fibroblastos, osteoblastos, condrócitos, células mesenquimais e células T e B ativadas e sua ligação com o seu receptor RANK (Receptor activator of NF-kB) em células precursoras de osteoclastos é necessário e suficiente para a ativação, diferenciação e sobrevivência de osteoclastos maduros. OPG, a outra proteína envolvida nesta modulação, serve como um falso receptor para RANKL, impedindo dessa forma a ligação RANKL-RANK e levando a uma menor ativação de osteoclastos. Assim, o balanço entre RANKL e OPG é o atual paradigma para a modulação da remodelação óssea. FK-506 (tacrolimo) é uma droga imunossupressora usada para prevenir rejeição de enxertos afetando a ativação de linfócitos T por meio da modulação da via da calcineurina, inibindo a ativação de NFAT e de NF-kB. Estudos prévios demonstraram que o uso de tacrolimo em ratos diminui a resposta inflamatória e reabsorção óssea em modelo experimental de indução de doença periodontal. A proposta deste estudo foi avaliar os efeitos da administração sistêmica do tacrolimo sobre a expressão de RANKL e OPG na doença periodontal induzida em ratos e determinar in vitro, se o tratamento de células residentes do periodonto com tacrolimo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Periodontitis is a well-characterized infectious disease with a complex immune-inflammatory response. In response to the bacterial presence, many resident and non-resident cells into the peridontium produce many cytokines and biologic mediators, causing tissue destruction and alveolar bone loss. These cytokines are the key-factor in osteoclast-mediated bone resorption. The expression ratio between two cytokines is fundamental to bone resorption process: RANKL (Receptor activator of NF-kB ligand) that is necessary to osteoclast differentiation, activation and survival, and OPG (Osteoprotegerin) that acts as the endogenous inhibitor of RANKL by functioning as its decoy receptor. RANKL is expressed by fibroblasts, osteoblasts, chondrocytes, mesenchymal cells and T and B lymphocytes. OPG is secreted primarily by osteoblastic cells, bone marrow stromal celss and fibroblasts and it counter regulates the excessive bone loss antagonizing the RANKL-binding to its receptor RANK in osteoclast precursor cells. The ratio between RANKL and OPG is the current paradigm for modulation of coupled bone turnover. FK-506 is an immunossupressive drug used to reduce and to prevent the risk of organ transplant rejections. It acts affecting T lymphocyte activation by calcinaurin pathway modulation inhibiting NFAT and NF-kB translocation to the nucleus. Previous studies showed that animals with experimental periodontitis treated with FK-506 exhibited less bone resorption and inflammatory infiltrate. The purpose of this study was evaluated effects of FK-506 systemic administration over RANKL and OPG expression in animals with experimental periodontitis; and determines if FK-506-treated periodontium resident cells can affect IL-1- and LPS-induced RANKL and OPG expression. In the in vivo study, two experimental periodontitis models were used (LPS and ligature) in rats. In the test group the animals received dail... (Complete abstract click electronic access below) / Doutor

O presente estudo teve como objetivo avaliar a eficácia do imunossupressor tópico tacrolimus a 0,1%, aplicado de forma oclusiva com o auxílio de moldeiras individualizadas de silicone em pacientes com gengivite descamativa decorrentes do líquen plano oral (LPO) e do penfigóide das membranas mucosas (PMM). Foram selecionados consecutivamente 18 pacientes que preencheram os critérios de inclusão adotados, sendo 17 mulheres (8 LPO / 9 PMM) e 1 homem com LPO. Após o estabelecimento do diagnóstico os pacientes foram avaliados por um mesmo avaliador quanto aos sinais e sintomas, utilizando-se para isso duas escalas visuais analógicas, uma para dor e outra para ardência, além de um periograma especialmente desenhado para avaliação clínica, demarcando as áreas onde haviam a presença de lesões gengivais no dia zero e no dia 90. Após adequação periodontal os pacientes eram moldados para confecção de uma moldeira individual de silicone para servir de suporte oclusivo para a medicação estudada. A terapêutica adotada foi dividida em duas fases de 45 dias totalizando 90 dias. Na primeira fase os pacientes foram orientados a usar de 1 a 2 gramas da medicação em cada moldeira, duas vezes ao dia por 20 minutos e na segunda fase somente uma vez ao dia. Os possíveis efeitos colaterais eram monitorados durante as consultas de retorno a cada 15 dias. Ao final dos 90 dias avaliamos o percentual de remissão dos sinais, classificada como completa (100%), excelente (75% a 99%), boa (50% a 74%), regular (1 a 44%), inalterada ou com piora do quadro. Nos pacientes estudados obtivemos remissão completa em 4 (30,76%), 4 com remissão excelente (30,76%), 4 com remissão boa (30,76%) e um com remissão regular (7,69%). Em relação aos sintomas da dor obtivemos uma redução média de 60% e em relação a ardência uma redução média de 65,5%. Os pacientes com LPO obtiveram uma redução média da dor de 42,5% e da ardência de 58%, nos pacientes com PMM a redução da dor foi de 92,8% e da ardência de 80,7%. A comparação dos escores de dor antes e após o tratamento apresentou valor de p<0,01 quando realizado o teste de Wilcoxon para amostras pareadas. O mesmo resultado (p<0,01) foi encontrado quando comparados os escores de ardência. Os valores obtidos demonstraram que a diminuição dos escores de dor e ardência foi estatisticamente significante. Como efeitos colaterais observamos que todos relataram alteração transitória de paladar e um paciente com LPO desenvolveu candidose. Concluímos que o tacrolimus a 0,1% aplicado topicamente com moldeiras individuais de silicone foi eficaz no tratamento das gengivites descamativas quanto a dor, ardência e remissão clínica das lesões gengivais em pacientes com LPO e PMM. / The objective of this study was to evaluate the efficacy of application of the topical immunosuppressor 0.1 % tacrolimus in patients with desquamative gingivitis (DG) associated with oral lichen planus (OLP) and mucous membrane pemphigoid (MMP). We selected 18 patients that fulfilled the inclusion criteria: 17 females (8 OLP/ 9MMP) and one male (OLP). After diagnosis, all subjects were evaluated by the same researcher, who assessed pain and burning using two visual analogical scales, one for pain and the other for burning, in conjunction with a diagram of the gingival mucosa. Measurements were made on day zero and day 90. After the periodontal assessment, individualized silicone rubber trays were prepared in order to deliver the drug. The therapeutic strategy was divided in two phases of 45 days for a total of 90 days. In the first treatment phase it was recommended that all patients use one or two grams of 0.1 % tacrolimus in his/her tray for twenty minutes, twice daily; in the second phase, the treatment was to be used only once daily. All side effects were monitored during biweekly return visits. At the end of 90 days, the response to therapy was assessed according to the following scale, as a percentage of remission: complete (100 %), excellent (75 % to 99 %), good (50 % to 74 %), poor (1 % to 49 %), no response (0 %) and worsened. We observed complete remission in 4 patients (30.76 % of the experimental group), excellent in 4 patients (30.76 %), good in 4 patients (30.76 %), and poor in one (7.69%). Pain was reported to be reduced by 60 % while burning was reduced by 65.5 %. OLP patients showed an average reduction by 42.5% of pain and 58% of burning. MMP patients showed an average reduction by 92.8% of pain and 80.7% of burning. After treatment, the data were analyzed by Wilcoxons test; significant differences (p < 0.01) were found for both pain and burning. Side effects included transitory alteration of taste, and one patient was diagnosed with candidiasis. From this study, we conclude that topical application of 0.1% tacrolimus using silicone rubber trays is an effective treatment for desquamative gingivitis, decreasing pain, burning and clinical lesions in OLP and MMP patients.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-05T14:19:45Z No. of bitstreams: 1 joaopaulomarinhoderezende.pdf: 4196573 bytes, checksum: ebdd159522bdab6897215b5587c36b68 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-10-06T12:29:39Z (GMT) No. of bitstreams: 1 joaopaulomarinhoderezende.pdf: 4196573 bytes, checksum: ebdd159522bdab6897215b5587c36b68 (MD5) / Made available in DSpace on 2016-10-06T12:29:39Z (GMT). No. of bitstreams: 1 joaopaulomarinhoderezende.pdf: 4196573 bytes, checksum: ebdd159522bdab6897215b5587c36b68 (MD5) Previous issue date: 2009-02-18 / O Líquen Plano Oral (LPO) é considerado uma doença inflamatória auto imune de curso crônico e sua presença pode estar relacionada com o aumento do estresse emocional. A sua relevância clínica está na possibilidade, ainda controversa, de se transformar em um carcinoma de células escamosas e de sua etiologia ainda obscura e desconhecida. Esse trabalho se propôs a tratar lesões de LPO que foram recalcitrantes aos tratamentos convencionais com corticosteróides, utilizando-se o Tacrolimo 0,1% (Protopic®) de uso tópico, aplicado duas vezes ao dia, por um período de oito semanas. Foram selecionados quinze pacientes (n = 15), que preencheram uma ficha de anamnese e uma escala visual de dor antes e depois do tratamento. Todos os pacientes se submeteram a uma biópsia inicial para o diagnóstico da doença e outra final para avaliar a ação da medicação sobre o infiltrado inflamatório. Foi realizado controle semanal, observando-se os aspectos clínicos, a sintomatologia dolorosa e a ocorrência de efeitos colaterais que quando presentes foram leves e transitórios. A análise dos resultados mostrou doze pacientes (80%) com remissão completa ou quase que completa das lesões e da sintomatologia dolorosa, dois pacientes (13,33%) apresentaram as lesões mais claras e apenas um paciente (6,67%) não obteve nenhuma alteração clínica e da sintomatologia dolorosa. Quanto à análise da regressão das características histológicas do LPO, notamos uma regressão moderada ou acentuada em doze pacientes (80%) e ausente ou leve em três pacientes (20%). Esses resultados nos fazem concluir que o Tacrolimo 0,1% (Protopic®) é uma medicação eficaz e segura, que melhora o aspecto clínico da lesão, diminui a sintomatologia dolorosa e reduz as características histopatológicas do LPO. / The oral lichen planus (OLP) has been considered a chronic-course auto immune inflammatory disease and its presence may be related to increased emotional stress. The clinical relevance of OLP is the possibility, in developing a squamous cell carcinoma which its etiology is still obscure and unknown. The aim of the present study is to treat OLP lesions that were recalcitrant to conventional treatment with corticosteroids, using topical Tacrolimus 0.1% (Protopic®), applied twice a day, for a period of eight weeks. Fifteen patients were selected who had filled out a history form and a visual scale for pain before and after treatment. All patients were submitted to an initial biopsy for the diagnosis of the disease and another one at the end to evaluate the effect of medication on the infiltrate. A weekly control was carried out, observing the clinical aspects, painful symptoms and the occurrence of side effects which, where present, were mild and transient. The results showed twelve patients (80%) with total or nearly total remission of painful symptoms and lesions, two patients (13.33%) showed the clearer lesions and only one patient (6.67%) did not obtain any change in clinical symptoms and pain. Throught the analysis of the histopathological features regression of OLP, if could be noticed a moderate or accented regression in twelve patients (80%) and an absent or mild regression in three patients (20%). Based on the our results, it was possible to conclude that the Tacrolimus 0.1% (Protopic®)is a safe and effective medication that improves the clinical aspect of the injury, reduces the painful symptoms and reduces the histopathological features of the OLP.

Introdução: Os imunossupressores das classes inibidores da calcineurina (tacrolimo) e da rapamicina (sirolimo) requerem controle terapêutico por apresentarem grande variabilidade farmacocinética que tem sido atribuída a fatores genéticos, entre outros. Poucos estudos avaliaram a expressão de genes alvo de imunossupressores e sua relação com a resposta terapêutica. Objetivo: Estudar a relação entre o perfil de expressão de genes alvos de tacrolimo e sirolimo e de microRNAs reguladores e a resposta à imunossupressores utilizados na profilaxia de rejeição ao transplante renal. Métodos: Participaram deste estudo 37 indivíduos submetidos ao transplante renal, no Hospital do Rim e Hipertensão da UNIFESP, de ambos os sexos, com idade acima de 18 anos e de qualquer etnia. Os pacientes foram tratados com esquema imunossupressor contendo tacrolimo, micofenolato de sódio e prednisona até o 3º mês quando foram randomizados para manter a terapia inicial (grupo TAC) ou para a conversão para sirolimo (grupo SRL). Os pacientes com disfunção renal ou suspeita de rejeição, no 3º mês, seguiram o tratamento inicial e foram avaliados em separado (grupo TACex). Os parâmetros de função renal e concentração sanguínea dos fármacos foram utilizados para monitoramento da terapia. A expressão de mRNA de MTOR, PPP3CA, PPP3CB, FKBP1A, FKBP1B e FKBP5, em leucócitos do sangue periférico, foi analisada por PCR em tempo real; e a expressão de miR-99a, miR-100, miR-145, miR-30a, miR-10b e miR-103a foi avaliada por PCR Array. Resultados: No primeiro mês de tratamento, a expressão diferencial de mRNA de MTOR, PPP3CA e FKBP1B diminuiu em relação ao pré-transplante (pre-Tx) (p<0,05). Esse efeito se manteve, no 3º mês, para MTOR e PPP3CA (p<0,05). A expressão diferencial de PPP3CB, FKBP1A e FKBP5 não foi alterada pelos tratamentos (p>0,05). No 6º mês, os grupos TAC, TACex e SRL apresentaram perfil de expressão diferencial de mRNA similar à do pre-Tx (p>0,05). No 3º mês, foram encontradas correlações positivas entre a expressão relativa de PPP3CB e creatinina sérica (r=0,49, p=0,04), e entre a expressão de FKBP1A e ureia (r=0,49, p=0,04), HDL colesterol (r= 0,53; p=0,02) e triglicérides (r=0,68, p=0,003) no soro. O perfil de expressão relativa de mRNA não se correlacionou com a concentração sanguínea de tacrolimo (p>0,05). Houve redução na expressão diferencial de miR-99a, no 3º mês, comparado com o pre-Tx (p<0,05) e a dos outros miRNAs não foi alterada. Não foi observada correlação entre o perfil de expressão relativo de miRNAs e mRNAs alvo, no 3º mês (p>0,05). Conclusão: A expressão diferencial de mRNA de MTOR, PPP3CA e FKBP1B e de miR-99a que tem como alvo o mRNA de MTOR, em leucócitos do sangue periférico, é modulada pelo tratamento imunossupressor a base de tacrolimo. A expressão diferencial de genes da via da calcineurina e do mTOR é sugestiva de sua potencial aplicação no monitoramento da terapia a base de tacrolimo. / Background: Immunosuppressive classes like calcineurin inhibitors (tacrolimus) and rapamycin (sirolimus) require therapeutic control because they have great pharmacokinetic variability that has been attributed to genetic factors, among others. Few studies have evaluated the expression of immunosuppressive target genes and their relation to therapeutic response. Objective: To study the relationship between the gene expression profile of tacrolimus and sirolimus targets and regulatory microRNAs with the response to immunosuppressive agents used in the prophylaxis of renal transplant rejection. Methods: This study included 37 patients undergoing kidney transplantation at the Hospital do Rim e Hipertensao/UNIFESP, age over 18 year old, both gender and any ethnicity. Patients were treated with an immunosuppressive regimen containing tacrolimus, mycophenolate sodium and prednisone during 3 months, when they were randomized to maintain the initial therapy (TAC group) or to convert to sirolimus (SRL group). Patients with renal dysfunction or signals of rejection in the 3rd month followed the initial treatment and were evaluated separately (TACex group). Parameters of renal function and blood concentration of immunosuppressive drugs were used to monitor therapy. The mRNA expression of MTOR, PPP3CA, PPP3CB, FKBP1A, FKBP1B and FKBP5 in peripheral blood leukocytes was analyzed by real-time PCR; and the expression of miR-99a, miR-100, miR-145, miR-30a, miR-10b and miR-103a was evaluated by PCR array. Results: In the first month of treatment, the differential mRNA expression of MTOR, PPP3CA and FKBP1B decreased relative to pre-transplant (pre-Tx) (p <0.05). This effect was maintained up to 3 month to MTOR and PPP3CA (p <0.05). The differential expression of PPP3CB, FKBP1A and FKBP5 was not affected by treatments (p> 0.05). On the 6th month, the TAC groups, TACex and SRL showed differential expression profile of mRNA similar to the pre-Tx (p> 0.05). On the 3rd month, positive correlations were found between the relative expression PPP3CB and serum creatinine (r =0.49, p =0.04) and between the expression of FKBP1A and urea (r=0.49, p=0.04), HDL cholesterol (r=0.53; p=0.02) and triglycerides (r = 0.68, p = 0.003) in serum. The relative mRNA expression profile was not correlated with tacrolimus blood concentrations (p> 0.05). There was a reduction in the differential expression of miR-99a, on the 3rd month, compared to the pre-Tx (p <0.05) and the other miRNAs has not changed. There was no correlation between the expression profile of miRNAs and mRNAs on target, on the 3rd month (p> 0.05). Conclusions: The differential expression of mRNA of MTOR, PPP3CA and FKBP1B and miR-99a which targets MTOR mRNA in peripheral blood leukocytes, is modulated by the immunosuppressant tacrolimus treatment base. The differential expression of genes of the calcineurin and mTOR is suggestive of their potential application in monitoring therapy tacrolimus base.

Le transfert adoptif de lymphocytes T virus-spécifiques ou ‘’Chimeric Antigen Receptors’’ (CAR) s’est avéré efficace pour le traitement de plusieurs types d’infections virales et certains cancers à la suite d’une greffe de cellules souches hématopoïétiques. Cependant, l’immunosuppression administrée pour la prévention du rejet de greffe et de la maladie du greffon contre l’hôte limite l’efficacité et la persistance à long terme des réponses médiées par ces lymphocytes. L’agent immunosuppresseur Tacrolimus (FK506) est parmi les plus utilisés, et fonctionne en liant la protéine FK506-Binding protein (FKBP12) afin d’exercer ses effets immunosuppresseurs sur les lymphocytes T. Dans le but de fournir une protection anti-virale, mais également anti-lymphoprolifératif des lymphocytes B et permettre la poursuite de cette immunosuppression préventive, nous avons pour objectif de générer des lymphocytes CAR-T et virus-spécifiques résistants à l’action du FK506 par l’invalidation du FKBP12. En utilisant la méthode d’édition génique basée sur les CRISPR ciblés par la nucléase Cas9, nous avons pu invalider le gène du FKBP12 sur des lymphocytes T stimulés par CD3-CD28. L’efficacité du knockout a été validée par Western Blot et TIDE sequencing. Le knock-out du gène du FKBP12 a conféré un maintien de la croissance cellulaire et des fonctions effectrices telles que la synthèse de cytokines IL-2, TNFα et IFN γ en présence de Tacrolimus comparativement aux cellules contrôles. Par la même méthode, nous avons pu invalider le gène du FKBP12 sur des lymphocytes T multivirus-spécifiques dont l’efficacité a été validée par cytométrie en flux. Les fonctions effectrices ont également été maintenues en présence de tacrolimus et ont été évaluées par ELISpot. Enfin, des lignées de lymphocytes T multivirus spécifiques dont le gène du FKBP12 a été invalidé ont été transduites avec un vecteur lentiviral dans le but d’exprimer un CAR CD19 dont l’expression a été validée par cytométrie en flux et la réactivité maintenue en présence de tacrolimus. En conclusion, ces résultats nous ont permis de démontrer la faisabilité de génération de lymphocytes T « triple fonction » anti-tumorales, anti-virales et résistantes au tacrolimus. L’application de cette approche semble prometteuse dans un contexte d’une immunothérapie adoptive anti-virale et anti-tumorale post-transplantation de moelle osseuse. / Adoptive transfer of virus-specific T lymphocytes or CAR-T cells has been shown to be effective for the treatment of several types of viral infections and certain cancers following hematopoietic cell transplantation. However, immunosuppression administered for the prevention of transplant rejection and graft-versus-host disease limits the efficacy and long-term persistence of responses mediated by these lymphocytes. The widely used immunosuppressive agent Tacrolimus (FK506) requires FK506-Binding protein (FKBP12) to exert its immunosuppressive effects on T cells. We undertook to engineer a multifunctional T-cell therapy to both optimally prevent viral reactivation and relapse of B-cell malignancies post-transplant in the context of immunosuppression. The objective of our work is to generate tacrolimus resistant, multivirus-specific T-cell lines expressing an anti-CD19 CAR. Using the gene editing method based on Clustered Regular interspaced short palidromic repeats (CRISPR) targeted by the CRISPR-associated protein 9 (Cas9) nuclease, we were able to invalidate the FKBP12 gene on activated T cells (confirmed by TIDE sequencing and western blotting). Invalidation of FKBP12 conferred maintenance of cell growth and effector functions such as the synthesis of cytokines IL-2, TNFα and IFNγ in the presence of Tacrolimus. Using the same method, we were able to delete the FKBP12 gene in virus-specific T lymphocytes. Effector functions were also maintained in the presence of tacrolimus. Finally, we integrated an anti-CD19 CAR by lentiviral transduction into FKBP12-edited multi-virus T-cell lines, and the efficiency of transduction was determined by flow cytometry. The cells maintained their viral reactivity in the presence of tacrolimus. In conclusion, we were able to confirm the feasibility of generation of ‘’triple function’’ T cells (anti-viral, anti-tumoral and tacrolimus resistant). Multifunctional T-cell product manufacturing is a promising approach to optimize post-transplant T-cell immunity against opportunistic pathogens and underlying malignancies.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Le récepteur neurokinine 1 (NK1R) est impliqué dans la régulation des réponses immunitaires innées et adaptatives. Cependant, les mécanismes par lesquels le NK1R modulerait ces réponses ne sont pas connus. Chez les cellules T, les voies de la calcineurine et de la mTOR constituent les cibles d’immunosuppresseurs, comme la cyclosporine A (CsA), le tacrolimus et la rapamycine. Ainsi, nous avons voulu déterminer si le NK1R pourrait agir sur ces voies et si le blocage pharmacologique du NK1R avec des antagonistes sélectifs, pourrait augmenter l’action de ces immunosuppresseurs sur l’activation des cellules T. Tout d’abord, nos résultats ont montré que les cellules Jurkat (celules T humaines) exprimaient à la fois le gène du NK1R et de son ligand (les endokinines). Ceci suggère l'existence d'une régulation autocrine tachykinergique de la fonction des cellules T. Cette hypothèse est appuyée par nos données, où nous avons observé que le blocage du NK1R avec des antagonistes spécifiques (L-733,060 et L-703,606) chez les cellules Jurkat, inhibe la production d'IL-2 et diminue l'activation du NFAT (substrat de la calcineurine). De façon intéressante, nous avons montré un effet de combinaison entre les antagonistes du NK1R et les inhibiteurs de la calcineurine (CsA et tacrolimus) sur la production d’IL-2 et l’activation du NFAT. En revanche, le blocage du NK1R n'a pas d'effet inhibiteur sur l’activation de la mTOR et la p70S6K, mais réduit la phosphorylation de S6R (Ser235/236) et Akt (Ser473). Enfin, nous n’avons observé aucun effet de combinaison avec la rapamycine et l’antagoniste NK1R sur l’activation de mTOR et de sa voie de signalisation. L’ensemble de nos résultats, démontrent la présence d'un nouveau mécanisme de régulation de NFAT impliquant le système tachykinergique NK1R/endokinines chez les cellules T. Par conséquent, nous suggérons que la combinaison des antagonistes NK1R avec les inhibiteurs de la calcineurine pourrait être une alternative thérapeutique intéressante afin de réduire les doses de CsA et le FK506 dans les protocoles de prévention de rejet de greffes. / There are increasing evidences for a role of the neurokinin 1 receptor (NK1R) in the regulation of innate and adaptive immune systems. However, whether NK1R regulates calcineurin/nuclear factor of activated T cell (NFAT) and mTOR pathways in T cells is unknown. These signalling pathways being targets of the immunosuppressive drugs cyclosporine A (CsA), FK506 and rapamycin respectively. We also examined whether pharmacological blockade of NK1R may be combined to those immunosuppressors to repress T cell activation. In this article, we first show that Jurkat T cells express both genes for NK1R and its ligands endokinins which suggests the existence of an autocrine tachykinergic regulation of T cells function. This hypothesis is supported by our data showing that blockade of this receptor with specific NK1R antagonists inhibits IL-2 production in Jurkat T cells which is associated with the reduction of NFAT activation. Interestingly, we show interplay between NK1R antagonists and calcineurin inhibitors to repress IL-2 production and NFAT activation. In contrast, blockade of NK1R has no inhibitory effect on mTOR and p70S6K activation but reduce S6R (Ser235/236) and Akt (Ser473) phosphorylation. However, combining rapamycin with NK1R antagonist has no enhancing effect on rapamycin-reduced mTOR activation and its signalling pathway. Our findings provide the evidence of a novel mechanism of regulation of NFAT activation-induced IL-2 production in T cells involving the tachykinergic system NK1R/endokinins. These observations may offer new application for NK1R antagonists in transplantation immunotherapy in combination with immunosuppressors.

L’immunosuppression optimale après greffe d’organe solide est une balance délicate et propre à chaque individu entre le risque de rejet et les risques liés à une surexposition au traitement immunosuppresseur. L’évaluation de la fonction résiduelle des lymphocytes T après stimulation par un mitogène (pharmacodynamie effective) devrait permettre de mesurer l’effet direct des médicaments immunosuppresseurs sur leur cible. Nous avons étudié différents paramètres de pharmacodynamie effective chez 34 receveurs pédiatriques de greffe d’organes solides traités par tacrolimus et mycophénolate. Les tests proposés dans ce travail sont adaptés au milieu pédiatrique et à une réalisation en temps réel. La quantification du CD25 parmi les CD4 activés par l’OKT3 permet de distinguer deux groupes de patients selon leur degré d’immunosuppression. L’âge médian est plus bas et la concentration plasmatique médiane en MPA plus élevée dans le groupe de patients plus fortement immunosupprimés. L’étude des paramètres immunologiques pouvant influencer la réponse (sécrétion des interleukines, proportion des sous-populations lymphocytaires CD4, CD8, T naïfs et Trég) ainsi que l’étude du pouvoir de restauration de la fonction lymphocytaire par l’Il-2, la guanosine ou la xanthosine, ne permettent pas de mieux comprendre les variabilités interindividuelles observées. Ces résultats devront être confirmés sur une cohorte plus grande de patients afin de juger de leur intérêt en pratique clinique. / Optimal immunosuppression following solid organ transplantation is unique to each individual and requires a balance between risks of rejection and overexposure to immunosuppressive therapy. The evaluation of residual function of T lymphocytes after mitogen stimulation (effective pharmacodynamic monitoring) should allow measurement of the direct effect of immunosuppressive drugs on their target. We studied various parameters of effective pharmacodynamic monitoring in 34 paediatric patients receiving solid organ transplants and treated with tacrolimus and mycophenolate (MPA). The tests proposed in this work are adapted to the paediatric setting in real time. Quantification of CD25 among CD4 cells activated by OKT3 can differentiate two groups of patients according to their degree of immunosuppression. Median values for age and MPA plasma concentration are lower and higher, respectively, in the patient group most heavily immunosuppressed. Neither study of the parameters that may influence the response (secretion of interleukins, proportion of lymphocyte subpopulations CD4, CD8, naive and regulatory T cells) nor study of the restoration of basal cell function brought about by Il2, guanosine or xanthosine, helped to explain the observed inter-individual variability. These results should be confirmed in a larger cohort of patients in order to test their relevance in clinical practice.

Ce travail de thèse porte sur l’application de la pharmacocinétique de population dans le but d’optimiser l’utilisation de certains médicaments chez les enfants immunosupprimés et subissant une greffe. Parmi les différents médicaments utilisés chez les enfants immunosupprimés, l’utilisation du busulfan, du tacrolimus et du voriconazole reste problématique, notamment à cause d’une très grande variabilité interindividuelle de leur pharmacocinétique rendant nécessaire l’individualisation des doses par le suivi thérapeutique pharmacologique. De plus, ces médicaments n’ont pas fait l’objet d’études chez les enfants et les doses sont adaptées à partir des adultes. Cette dernière pratique ne prend pas en compte les particularités pharmacologiques qui caractérisent l’enfant tout au long de son développement et rend illusoire l’extrapolation aux enfants des données acquises chez les adultes. Les travaux effectués dans le cadre de cette thèse ont étudié successivement la pharmacocinétique du busulfan, du voriconazole et du tacrolimus par une approche de population en une étape (modèles non-linéaires à effets mixtes). Ces modèles ont permis d’identifier les principales sources de variabilités interindividuelles sur les paramètres pharmacocinétiques. Les covariables identifiées sont la surface corporelle et le poids. Ces résultats confirment l’importance de tenir en compte l’effet de la croissance en pédiatrie. Ces paramètres ont été inclus de façon allométrique dans les modèles. Cette approche permet de séparer l’effet de la mesure anthropométrique d’autres covariables et permet la comparaison des paramètres pharmacocinétiques en pédiatrie avec ceux des adultes. La prise en compte de ces covariables explicatives devrait permettre d’améliorer la prise en charge a priori des patients. Ces modèles développés ont été évalués pour confirmer leur stabilité, leur performance de simulation et leur capacité à répondre aux objectifs initiaux de la modélisation. Dans le cas du busulfan, le modèle validé a été utilisé pour proposer par simulation une posologie qui améliorerait l’atteinte de l’exposition cible, diminuerait l’échec thérapeutique et les risques de toxicité. Le modèle développé pour le voriconazole, a permis de confirmer la grande variabilité interindividuelle dans sa pharmacocinétique chez les enfants immunosupprimés. Le nombre limité de patients n’a pas permis d’identifier des covariables expliquant cette variabilité. Sur la base du modèle de pharmacocinétique de population du tacrolimus, un estimateur Bayesien a été mis au point, qui est le premier dans cette population de transplantés hépatiques pédiatriques. Cet estimateur permet de prédire les paramètres pharmacocinétiques et l’exposition individuelle au tacrolimus sur la base d’un nombre limité de prélèvements. En conclusion, les travaux de cette thèse ont permis d’appliquer la pharmacocinétique de population en pédiatrie pour explorer les caractéristiques propres à cette population, de décrire la variabilité pharmacocinétique des médicaments utilisés chez les enfants immunosupprimés, en vue de l’individualisation du traitement. Les outils pharmacocinétiques développés s’inscrivent dans une démarche visant à diminuer le taux d'échec thérapeutique et l’incidence des effets indésirables ou toxiques chez les enfants immunosupprimés suite à une transplantation. / This thesis deals with the application of population pharmacokinetics in order to optimize the use of certain medications in immunocompromised children undergoing transplantation. Among the various drugs used in immunocompromised children, the use of busulfan, tacrolimus and voriconazole remains problematic, particularly because of high interindividual variability in their pharmacokinetics necessitating individualized doses based on therapeutic drug monitoring. In addition, these drugs have not been studied in children and the doses are adapted from adults. This practice does not take into account the pharmacological characteristics of pediatrics throughout their development and makes illusory the extrapolation of data acquired in adults to children. The work done in this thesis studied sequentially the pharmacokinetics of busulfan, voriconazole and tacrolimus by a population approach (non-linear mixed effects models). The developed models have identified the main sources of interindividual variability in the pharmacokinetic parameters of these drugs. The identified covariates are body surface area and weight. These results confirm the importance of taking into account the effect of growth in children. These parameters were allometrically included in the models. This approach allows separating the effect of size from other covariates and enables the comparison of pediatric pharmacokinetic parameters with those of adults. The inclusion of these explanatory covariates should improve the management a priori of patients. The developed models were evaluated to confirm their stability, performance, and their ability to answer the original objectives of modeling. In the case of busulfan, the validated model was used to simulate dosing regimens that improve reaching the target exposure, reduce treatment failure and toxicity episodes. The developed population pharmacokinetic model for voriconazole confirmed the large variability in its pharmacokinetics in immunocompromised children. The limited data did not allow identification of covariates explaining this variability. Based on the population pharmacokinetic model of tacrolimus, a Bayesian estimator was developed, which is the first in this population of pediatric liver transplant recipients. This estimator can predict pharmacokinetic parameters and individual exposure to tacrolimus based on a limited number of samples. In conclusion, this thesis allowed applying the population pharmacokinetics approach in pediatrics to explore the characteristics of this population and describe the pharmacokinetic variability of drugs used in immunocompromised children, for the individualization of treatment. Pharmacokinetic tools developed are part of efforts to decrease the rate of treatment failure and the incidence of adverse and toxic events in immunocompromised and transplanted pediatrics.

Para avaliar a exposição ao ácido micofenólico (MPA) na fase inicial pós-transplante renal, receptores foram destinados para receber tacrolimo (n=33) ou ciclosporina (n=19, controle) com MMF. Foram feitas coletas de farmacocinética (AUC) do inibidor de calcineurina e MPA nos dias 7, 14, 30, 60 e 180 pós-transplante. Dos dias 14-180, a MPA-AUC foi mais elevada no grupo tacrolimo devido a um maior segundo pico de MPA. Com doses fixas de MMF, uma grande porcentagem de curvas ficou abaixo da faixa terapêutica. No dia 7, a equação que emprega a concentração pré-dose (C0) e na segunda hora (C2) foi a mais precisa para estimar AUC. Após o dia 7, a equação que utiliza C2 foi a mais precisa. A exposição ao MPA nos primeiros seis meses após transplante renal é maior sob tacrolimo do que ciclosporina. Entretanto, para qualquer inibidor de calcineurina empregado com MMF, uma equação que emprega C0 e C2 (dia 7) e C2 isoladamente (após o dia 7), permite a monitoração de MPA com grande precisão / To evaluate the exposure to mycophenolic acid (MPA) early after renal transplantation, recipients were allocated to tacrolimus (n = 33) or Neoral (n =19, control) plus MMF. Pharmacokinetic curves (AUC) of calcineurin inhibitor and MPA were drawn on days 7, 14, 30, 60 and 180 post-transplant. From days 14-180, MPA-AUC was higher in tacrolimus group due to a higher second MPA peak. With fixed MMF doses, a great amount of curves fell below the proposed therapeutic range. On day 7, the equation that uses pre-dose (C0) and second-hour (C2) concentrations was the most accurate. After day 7, the equation that uses C2 alone was the most accurate. Exposure to MPA during the first six months after transplantation is higher under tacrolimus than Neoral. Nevertheless, despite the calcineurin inhibitor associated with MMF, an equation that uses C0 and C2 up to day 7 and C2 thereafter allows precise MPA monitoring