Background: Evidence-based treatment algorithms, successfully established for asthma, are missing for atopic eczema (AE). Objectives: To investigate whether treatment according to an evidence-based algorithm is an effective and applicable concept for the management of AE. Methods: Based on a systematic literature review, we developed an evidence-based severity-score-oriented treatment algorithm for AE and compared its effectiveness to that of an individualised symptom-oriented treatment (individual therapy) in a randomised controlled trial. Sixty-three participants were randomised to algorithm (n = 32) or individual therapy (n = 31) and treated accordingly for 12 months. Study end points included difference between baseline SCORAD and mean SCORAD under treatment (primary end point), quality of life and treatment utilisation. Analysis was by intention to treat (registration: ClinicalTrials.gov:NCT00148746). Results: No statistically significant differences in clinical or subjective response were observed between groups. Treatment following the algorithm and individual treatment both effectively controlled AE. Mean SCORAD reductions were 47% (95% confidence interval, CI = 38–55; algorithm) and 42% (95% CI = 29–54; individual). Clinical response was paralleled by improved quality of life in both groups. Physicians adhered to the algorithm option in 93% of their treatment decisions. Conclusion: Treatment following an evidence-based algorithm is an effective and applicable concept for the management of AE but does not show clear advantages compared to individualised treatment in a dermatological setting. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Herzinsuffizienz mit konsekutivem Herzversagen ist ein zentrales kardiovaskuläres Problem der heutigen Bevölkerung.Ursächlich ist insbesondere eine progrediente Herzhypertrophie. Die Calcium-Calmodulin abhängige Phosphatase Calcineurin (CnR) spielt hierbei in der Pathogenese eine entscheidende Rolle. CnR wird über seine Calciumbindungsstellen an der regulatorischen Untereinheit Calcineurin B (CnB) aktiviert.Um zu untersuchen, inwieweit CnB bei der Hypertrophie verschiedener Ätiologien reguliert wird, wurde in linksventrikulären Myokardbiopsien von Patienten mit Aortenstenose (AS= 14) bzw. aus explantierten Herzen mit Dilatativer Kardiomyopathie (DCM=27) und Koronarer Herzerkrankung (KHK=7) der mRNA-und Proteingehalt von CnB bestimmt und mit der Expression von ANP und BNP korreliert. Als Kontrollgruppe dienten 15 abgelehnte Spenderherzen mit normaler systolischer Funktion und gesunder Morphologie. In den Herzen der Kontroll-, DCM-, und KHK-Gruppen wurde der linksventrikuläre Fibrosegehalt bestimmt. Hierzu wurden eine extern standardisierte Real-Time-PCR-Technik und ein etabliertes Western Blot Verfahren angewandt. Die Ergebnisse werden im Median ± 25%/75%-Perzentile angegeben und mit dem Mann-Whitney-Test bzw. Korrelationsanalysen nach Spearman berechnet. In den Herzen mit DCM zeigte sich eine signifikante Erhöhung der CnB mRNA auf ca. das Dreifache der Kontrollen (293% der Ko, p / Heart failure is a central cardiovascular problem for the current population. Cardiac hypertrophy is a central factor. The calcium-Calmodulin dependent phosphatase Calcineurin (CnR) plays a crucial role in the pathogenesis. CnR is activated via its calcium-binding site in the regulatory subunit Calcineurin B (CnB). In order to examine, to what extent CnB is regulated in different aetiologies of hypertrophy, we analysed CnB´s mRNA and protein in left ventricular samples from patients with aortic valve stenosis (AS = 14) and from explanted hearts with dilated (DCM=27) and ischemic (ICM=7) cardiomyopathy and correlated them with the expression of ANP and BNP. As a control, 15 rejected donor hearts with normal systolic function and non-pathologic changed morphology were used. Fibrosis of the left ventricle was determined in three groups: control , DCM and ICM. Therefore, we used an externally standardized real-time PCR and an established Western Blot. Data are given as median ± 25%/75%- percentiles; Mann Whitney test and Spearman´s correlation-analyses were used. CnB mRNA was significantly raised in DCM (293% of control, p

Therapeutic approaches leading to the stimulation of regeneration, and/or inhibition of degeneration processes in neuromuscular disorders are believed to offer valid therapeutic strategies that would preserve muscle tone and contribute to the quality of life while lengthening patient life span. Activation of CalcineurinA (CnA), a threonine-serine phosphatase, controls gene regulatory programs in skeletal muscle by stimulating slow muscle fiber (type I) gene expression. This phosphatase has been also identified as a key mediator in the hypertrophic response and in skeletal muscle regeneration. Activation of CnA is, therefore, considered as a potentially interesting means of stimulating muscle regeneration in myopathies. We have identified a peptide aptamer that activates CnA in vitro, in cells and in vivo. In a mouse model for denervation-induced muscle atrophy, CnA-activating peptide aptamers show significant positive impact. This is reflected in larger overall muscle cross-sectional surface area due to an increased number of fibers and larger individual fiber surface area. Insight into the biological mechanism is afforded by observation of increased levels of nuclear NFAT transcription factor in these fibers, in agreement with peptide aptamer-mediated activation of CnA. Furthermore, a significant increase in central nuclei, characteristic of the presence of new fibers, is observed in muscles treated with the peptide aptamers specifically activating CnA. Identification of the specific binding site of the peptide aptamer on CnA was achieved using several truncations of the phosphatase, offering insight into the molecular mechanism of action. Together, these studies offer the first proof that direct activation of endogenous CnA has a measureable impact on cellular responses resulting in stimulation of muscle regeneration and enhancement of pathophysiological state in selected animal models.

Orientador: Luís Gustavo Modelli de Andrade / Resumo: Introdução: O Brasil é o maior serviço público de transplantes do mundo sendo a quase totalidade do seu financiamento custeado pelo sistema único de saúde. Os regimes de imunossupressão também são inteiramente financiados pelo Estado através do Componente Especializado de Assistência Farmacêutica, mas são pouco estudados sob a perspectiva econômica. O objetivo primário foi realizar uma análise de custo efetividade do regime de imunossupressão de tacrolimo com micofenolato comparado a tacrolimo associado a imTOR pelo período de um ano após o transplante renal. O objetivo secundário foi avaliar a não inferioridade destes dois regimes. Materiais e Métodos: Tratou-se de um estudo retrospectivo de transplantados renais realizados no Hospital das Clínicas de Botucatu, comparando protocolos de imunossupressão em 12 meses após o transplante. O primeiro esquema de imunossupressão foi o utilizado até outubro de 2015 e o segundo esquema adotado após mudança do protocolo do serviço. Foram feitas comparações entre os dois regimes de imunossupressão para avaliar a não inferioridade sendo definida como: o somatório dos eventos de rejeição aguda, descontinuidade, perda do enxerto e óbito ao fim de um ano. Para as análises de custo foi construído um modelo de análise de decisão no software TreeAge Pro 2009. As probabilidades de ocorrência dos eventos foram calculadas em cada grupo na amostra histórica. Foram conduzidas análises de sensibilidade visando obter uma simulação realista dos custos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Brazil is the largest public transplant service in the world, with almost all of its costs funded by the public health system. The immunosuppressive regimens are also fully funded by the state. These regimes were studied in terms of effectiveness, but they were poorly studied from an economic perspective. The primary objective was to conduct a cost-effectiveness analysis of the immunosuppressive regimen of tacrolimus with mycophenolate compared to tacrolimus associated with imTOR for a period of one year after kidney transplantation. The secondary objective was to assess the non-inferiority of these two regimens. Materials and Methods: This was a retrospective study of kidney transplants carried out at UNESP, Univ Estadual Paulista, comparing immunosuppressive protocols at 12 months after transplantation. The first immunosuppressive regimen was the one used until October 2015 and the second regimen adopted after changing the service protocol. Comparisons were made between the two immunosuppressive regimens to evaluate non-inferiority, defined as a composite end-point of biopsy proven acute rejection, graft loss, death, and adverse events leading to drug discontinuation. A decision analysis model was constructed with TreeAge Pro 2009 software to perform the cost analyzes. The probability of occurrence of clinical events was calculated for each group in the historical sample. Sensitivity analyzes were conducted in order to obtain a realistic simulation of the fina... (Complete abstract click electronic access below) / Mestre

Tacrolimus (TAC) é um fármaco imunossupressor muito utilizado na prevenção de rejeição aguda após o transplante de órgãos. Essa droga apresenta um índice terapêutico muito baixo e grande variabilidade intra e interindividual, sendo necessário programas de monitorização terapêutica para se otimizar a eficácia e limitar a toxicidade. O TAC é um fármaco substrato do CYP3A5 e transportado pela proteína de efluxo PGP e acredita-se que o polimorfismos genéticos (SNPs) destas proteínas estejam relacionados a alta variabilidade farmacocinética desta droga. Neste estudo, investigamos a influência dos polimorfismos destas proteínas sobre alguns parâmetros farmacocinéticos do TAC e também, na incidência de lesões renais e rejeição em receptores de transplante renal. Pacientes recebendo TAC a no mínimo 12 meses (n=108) foram genotipados (PCR real time) para os polimorfismos do CYP3A5*3 (rs776746) e do gene ABCB1 1236C>T (rs1128503), 2677G>T/A (rs2032582) e 3435C>T (rs1045642). Dados da concentração plasmática de vale (Co; ng/mL), dose diária normalizada (mg/dia por Kg do paciente) e a concentração plasmática do fármaco normalizada pela dose ingerida (Co/dose, ng/mL por mg/dia por kg do paciente) de TAC foram obtidos dos prontuários médicos ao longo de três anos após o transplante renal. O desfecho clínico foi analisado avaliando-se a curva de sobrevida o enxerto, a função renal obtida pelo clearance de creatinina (Equação de Cockroft-Gault), e desenvolvimento de lesões renais e rejeição aguda e crônica com diagnósticos estabelecidos mediante suspeita clínica e confirmados pela avaliação das biópsias quanto a presença de necrose tubular aguda (NTA) e nefropatia crônica do enxerto (NFC) de acordo com a classificação de BANFF 07. Os haplótipos do gene ABCB1 foram inferidos estatisticamente utilizando o software PHASE (version 2.1). Diferenças foram consideradas significativas quando p<0,05. Não observamos desvios em relação ao esperado pelo equilíbrio de Hardy-Weinberg em nossa população para nenhum dos genes estudados. Frequências alélicas destes polimorfismos (6986G 74%; 1236C 60%; 3435C 59% and 2677G 64%) e haplótipos (49% 2677G-3435C-1236C e 31% 2677T-3435T-1236T) foram consistentes com outros estudos realizados na população brasileira. Indivíduos portadores de ao menos um alelo *1 do gene CYP3A5 necessitavam de maiores doses de TAC para obter níveis plasmáticos semelhantes aos dos indivíduos homozigotos para o alelo *3 (0,09 ± 0,03 vs. 0,06 ± 0,03; mg/dia/kg; p<0,001). Ao final do primeiro ano de transplante pacientes CYP*3/*3 apresentavam praticamente o dobro da razão Co/dose quando comparados com os pacientes CYP*1/*1 (144,60 ± 67,29 vs. 70,44 ± 56,05 ng*mL-1 /mg*kg- 1 /dia; p<0,001). Observou-se semelhante em relação quando indivíduos portadores dos alelos e haplótipo variante da PGP foram avaliados. Não encontramos associações significativas entre os genótipos e a sobrevida do enxerto ou clearance de creatinina. No entanto, observamos que os pacientes portadores do haplótipo GCC apresentaram maior incidência de desenvolvimento de NFC. Este estudo confirma o efeito dos polimorfismos do CYP3A5 e, em menor grau da PGP na farmacocinética do TAC. No entanto, não encontramos associações entre esses polimorfismos e desfechos clínicos significantes, sugerindo que a genotipagem para o CYP3A5 ou ABCB1 ainda não deva ser incorporada na prática clínica como uma ferramenta para o manejo de transplante renal. / Tacrolimus (TAC) is widely used to prevent acute rejection following solid-organ transplantation. This drug is characterized by a narrow therapeutic index and drug monitoring programs are required both to optimize efficacy and to limit toxicity. TAC is known to be substrate of cytochrome P450 (CYP) 3A5 and P-glycoprotein (PGP/ABCB) and its been suggested that genetic polymorphisms (SNPs) of these proteins are highly associated with variations in TAC pharmacokinetics. We investigated the influence of polymorphisms of CYP3A5 and ABCB1 gene on the pharmacokinetic parameters (PK) of TAC and on the incidence of kidney injuries and allograft rejection (AR) in renal transplant recipients. Patients receiving TAC for at least 12 months (n=108) were genotyped (real-time PCR) for CYP3A5*3 (rs776746) and for ABCB1 1236C>T (rs1128503), 2677G>T/A (rs2032582) and 3435C>T (rs1045642) polymorphisms. TAC predose concentration (Co; ng/mL), TAC daily dose (mg/day per kg body weight) and dose-normalized predose concentrations (Co/dose; ng/mL per mg/day per kg body weight) were retrieved from medical records up to 03 years after transplantation. Clinical outcomes were analyzed evaluating renal function in terms of creatinine clearance ( Cockroft-Gault equation) and allograft survival. Kidney injuries and AR diagnostics were established by clinical suspicion and in presence of histological findings in renal biopsies according to the 2007 Banff classification. ABCB1 gene haplotypes were statistically inferred using PHASE software (version 2.1). No deviation from Hardy-Weinberg equilibrium was observed in our study population for the polymorphic loci examined in CYP3A5 and ABCB1. Allelic frequencies of these polymorphisms (6986G 74%; 1236C 60%; 3435C 60% and 2677G 65%) and haplotypes (49% 2677G-3435C-1236C and 30% 2677T- 3435T-1236T) were consistent with other studies in the Brazilian population. Individuals carrying at least one CYP3A5*1 allele required higher TAC dose to achieve similar TAC blood levels as the homozygous individuals for the *3 allele (0.09 ± 0.03 vs.0.06 ± 0.03, mg/day per kg body weight, p<0.001). The presence of the CYP3A5*1 allele was also associated with lower TAC Co/dose compared to CYP*3 homozygous (84.9 ± 43.2 vs. 144.6 ± 66.7 ng/mL per mg/day per kg body weight, p<0.001). Regarding ABCB1 polymorphisms, individuals homozygous for the variant allele of each individual SNP and for the haplotype (TTT) showed higher Co/dose ratio. No associations were found between SNPs or haplotypes and allograft survival or creatinine clearance. We did find, though, that patients carrying GCC haplotype had a higher incidence of chronic rejection. Our findings confirm the effect of CYP3A5 and, less pronounced of ABCB1 polymorphisms, on the TAC pharmacokinetic. On the other hand, we did not find any association between these polymorphisms and relevant clinical outcomes, suggesting that CYP3A5 and ABCB1 genotyping must not be incorporated as a useful clinical tool on the management of kidney transplantation.

Tacrolimus (TAC) é um fármaco imunossupressor muito utilizado na prevenção de rejeição aguda após o transplante de órgãos. Essa droga apresenta um índice terapêutico muito baixo e grande variabilidade intra e interindividual, sendo necessário programas de monitorização terapêutica para se otimizar a eficácia e limitar a toxicidade. O TAC é um fármaco substrato do CYP3A5 e transportado pela proteína de efluxo PGP e acredita-se que o polimorfismos genéticos (SNPs) destas proteínas estejam relacionados a alta variabilidade farmacocinética desta droga. Neste estudo, investigamos a influência dos polimorfismos destas proteínas sobre alguns parâmetros farmacocinéticos do TAC e também, na incidência de lesões renais e rejeição em receptores de transplante renal. Pacientes recebendo TAC a no mínimo 12 meses (n=108) foram genotipados (PCR real time) para os polimorfismos do CYP3A5*3 (rs776746) e do gene ABCB1 1236C>T (rs1128503), 2677G>T/A (rs2032582) e 3435C>T (rs1045642). Dados da concentração plasmática de vale (Co; ng/mL), dose diária normalizada (mg/dia por Kg do paciente) e a concentração plasmática do fármaco normalizada pela dose ingerida (Co/dose, ng/mL por mg/dia por kg do paciente) de TAC foram obtidos dos prontuários médicos ao longo de três anos após o transplante renal. O desfecho clínico foi analisado avaliando-se a curva de sobrevida o enxerto, a função renal obtida pelo clearance de creatinina (Equação de Cockroft-Gault), e desenvolvimento de lesões renais e rejeição aguda e crônica com diagnósticos estabelecidos mediante suspeita clínica e confirmados pela avaliação das biópsias quanto a presença de necrose tubular aguda (NTA) e nefropatia crônica do enxerto (NFC) de acordo com a classificação de BANFF 07. Os haplótipos do gene ABCB1 foram inferidos estatisticamente utilizando o software PHASE (version 2.1). Diferenças foram consideradas significativas quando p<0,05. Não observamos desvios em relação ao esperado pelo equilíbrio de Hardy-Weinberg em nossa população para nenhum dos genes estudados. Frequências alélicas destes polimorfismos (6986G 74%; 1236C 60%; 3435C 59% and 2677G 64%) e haplótipos (49% 2677G-3435C-1236C e 31% 2677T-3435T-1236T) foram consistentes com outros estudos realizados na população brasileira. Indivíduos portadores de ao menos um alelo *1 do gene CYP3A5 necessitavam de maiores doses de TAC para obter níveis plasmáticos semelhantes aos dos indivíduos homozigotos para o alelo *3 (0,09 ± 0,03 vs. 0,06 ± 0,03; mg/dia/kg; p<0,001). Ao final do primeiro ano de transplante pacientes CYP*3/*3 apresentavam praticamente o dobro da razão Co/dose quando comparados com os pacientes CYP*1/*1 (144,60 ± 67,29 vs. 70,44 ± 56,05 ng*mL-1 /mg*kg- 1 /dia; p<0,001). Observou-se semelhante em relação quando indivíduos portadores dos alelos e haplótipo variante da PGP foram avaliados. Não encontramos associações significativas entre os genótipos e a sobrevida do enxerto ou clearance de creatinina. No entanto, observamos que os pacientes portadores do haplótipo GCC apresentaram maior incidência de desenvolvimento de NFC. Este estudo confirma o efeito dos polimorfismos do CYP3A5 e, em menor grau da PGP na farmacocinética do TAC. No entanto, não encontramos associações entre esses polimorfismos e desfechos clínicos significantes, sugerindo que a genotipagem para o CYP3A5 ou ABCB1 ainda não deva ser incorporada na prática clínica como uma ferramenta para o manejo de transplante renal. / Tacrolimus (TAC) is widely used to prevent acute rejection following solid-organ transplantation. This drug is characterized by a narrow therapeutic index and drug monitoring programs are required both to optimize efficacy and to limit toxicity. TAC is known to be substrate of cytochrome P450 (CYP) 3A5 and P-glycoprotein (PGP/ABCB) and its been suggested that genetic polymorphisms (SNPs) of these proteins are highly associated with variations in TAC pharmacokinetics. We investigated the influence of polymorphisms of CYP3A5 and ABCB1 gene on the pharmacokinetic parameters (PK) of TAC and on the incidence of kidney injuries and allograft rejection (AR) in renal transplant recipients. Patients receiving TAC for at least 12 months (n=108) were genotyped (real-time PCR) for CYP3A5*3 (rs776746) and for ABCB1 1236C>T (rs1128503), 2677G>T/A (rs2032582) and 3435C>T (rs1045642) polymorphisms. TAC predose concentration (Co; ng/mL), TAC daily dose (mg/day per kg body weight) and dose-normalized predose concentrations (Co/dose; ng/mL per mg/day per kg body weight) were retrieved from medical records up to 03 years after transplantation. Clinical outcomes were analyzed evaluating renal function in terms of creatinine clearance ( Cockroft-Gault equation) and allograft survival. Kidney injuries and AR diagnostics were established by clinical suspicion and in presence of histological findings in renal biopsies according to the 2007 Banff classification. ABCB1 gene haplotypes were statistically inferred using PHASE software (version 2.1). No deviation from Hardy-Weinberg equilibrium was observed in our study population for the polymorphic loci examined in CYP3A5 and ABCB1. Allelic frequencies of these polymorphisms (6986G 74%; 1236C 60%; 3435C 60% and 2677G 65%) and haplotypes (49% 2677G-3435C-1236C and 30% 2677T- 3435T-1236T) were consistent with other studies in the Brazilian population. Individuals carrying at least one CYP3A5*1 allele required higher TAC dose to achieve similar TAC blood levels as the homozygous individuals for the *3 allele (0.09 ± 0.03 vs.0.06 ± 0.03, mg/day per kg body weight, p<0.001). The presence of the CYP3A5*1 allele was also associated with lower TAC Co/dose compared to CYP*3 homozygous (84.9 ± 43.2 vs. 144.6 ± 66.7 ng/mL per mg/day per kg body weight, p<0.001). Regarding ABCB1 polymorphisms, individuals homozygous for the variant allele of each individual SNP and for the haplotype (TTT) showed higher Co/dose ratio. No associations were found between SNPs or haplotypes and allograft survival or creatinine clearance. We did find, though, that patients carrying GCC haplotype had a higher incidence of chronic rejection. Our findings confirm the effect of CYP3A5 and, less pronounced of ABCB1 polymorphisms, on the TAC pharmacokinetic. On the other hand, we did not find any association between these polymorphisms and relevant clinical outcomes, suggesting that CYP3A5 and ABCB1 genotyping must not be incorporated as a useful clinical tool on the management of kidney transplantation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine involved in the production and function of hematopoietic cells, and GM-CSF plays in particular a major role in responses to infection and physiological and pathological inflammatory processes. GM-CSF is produced in many cell types, and increases in the intracellular Ca2+ concentration are, like in many other systems, of major importance in the intracellular signaling that determines GM-CSF expression after receptor stimulation of the cells. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) mediates stimulation of GM-CSF transcription in response to Ca2+. This thesis shows that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Mutation of the CaMK II target serines increased transactivation of the GM-CSF promoter/enhancer and decreased the sensitivity to inhibition by increased Ca2+ or constitutively active CaMK II. The Ca2+-dependent phosphorylation of Ets1 was also shown to reduce the binding of Ets1 to the GM-CSF promoter in vivo. RUNX1, also known as acute myeloid leukemia 1 (AML1), is one of three mammalian RUNX transcription factors and has many essential functions in hematopoiesis. RUNX1 has also many important roles in the immune system, and RUNX1 is the most frequent target for chromosomal translocation of genes in acute human leukemias. This thesis shows that RUNX1 directly interacts with both subunits of CN and that the strongest interaction is localised to the regulatory CN subunit and the DNA binding domain of the RUNX protein. Constitutively active CN was shown to activate the promoter/enhancer of GM-CSF synergistically with RUNX1, RUNX2 or RUNX3, and the Ets1 binding site of the promoter was shown to be essential for the synergy between RUNX1 and CN in Jurkat T cells. The analysis suggests that Ets1 phosphorylated by the protein kinase glycogen synthase kinase-3β is the target of RUNX1-recruited CN phosphatase at the GM-CSF promoter. Transforming growth factor-β (TGF-β) is another multipotent cytokine that often has a role opposite to that of GM-CSF in inflammatory responses since it is a potent suppressor of immune cells and therefore is anti-inflammatory. This thesis shows that TGF-β can decrease transcription from a GM-CSF promoter/enhancer. Certain constitutively active TGF-β receptors and the TGF-β activated transcription factor Smad3 could also repress GM-CSF transcription, whereas several other Smad proteins did not have this inhibitory effect. The inhibition required intact DNA binding ability of Smad3, and the 125 bp upstream of the transcription initiation site, which was sufficient for the inhibition, contains several weak Smad binding sites near the TATA box next to an Ets1 site of the promoter. Smad3 was able to bind to the promoter DNA together with Ets1 and could also be in complex with Ets1 in the absence of DNA. Surface plasmon resonance analysis revealed that Ets1 interacted with the DNA binding domain of Smad3, and the binding constant of this interaction was about 1 µM. The results identify a negative regulation of the GM-CSF promoter by TGF-β signaling through direct Smad3 binding and indicate that the mechanism is by Smad3 interaction with Ets1 and perhaps other proteins around the TATA box of the promoter. This thesis also identifies a novel transactivation domain in the N-terminal of RUNX1 including the N-terminal α-helix in the DNA binding domain. The domain was also required for RUNX2 and RUNX3 transactivation. Despite this, the N-terminal domain of RUNX1 was not essential for RUNX1 function in megakaryocytopoiesis in vitro from mouse embryonic stem cells.

Occupational exposure to endotoxin-rich swine barn air induces respiratory diseases and loss of lung function. Barn exposure induces recruitment of pulmonary intravascular monocytes/macrophages (PIMMs) and subsequent increased host sensitivity to <i>Escherichia coli</i> LPS challenge. Therefore, to further clarify the biology of PIMMs we examined the role of recruited PIMMs in a rat <i>Escherichia coli</i>-induced lung inflammation model. Following sepsis, lung inflammation was induced with recruitment of PIMMs and subsequently, <i>Escherichia coli</i> LPS challenge exacerbated the lung inflammation with localization of multiple inflammatory cytokines in PIMMs to suggest their possible involvement in modulating lung inflammation in this model.<p> In order to delineate mechanisms of barn air induced lung dysfunction, a rat model of occupational exposure was characterized to show that one and five exposures to the barn environment induced acute lung inflammation and increased airway hyperresponsiveness (AHR). Following 20 exposures, AHR was dampened to indicate adaptive responses. Barn air contains high levels of endotoxin which led us to investigate its role in lung inflammation and AHR. Exposure of mice with either a functional TLR4 (WT) or non-functional TLR4 (mutants) to barn air revealed dependence of lung inflammation but not AHR on a functional TLR4.<p> I investigated whether exposure to barn air alters host responses to a subsequent microbial challenge. Following one day barn exposure and <i>Escherichia coli</i> LPS challenge, lung inflammation was exacerbated with increased granulocytes and IL-1β levels compared to one day barn exposed rats without <i>Escherichia coli</i> LPS challenge. However, increased granulocytes and IL-1β levels in barn exposed and <i>Escherichia coli</i> LPS challenged rats were not different from control rats treated with <i>Escherichia coli</i> LPS indicating a lack of priming effect of barn exposure. However, above results are suggestive of an underlying risk of increased lung inflammation following secondary microbial infection in naïve barn workers.<p> Lastly, I investigated the expression and activity of novel signalling molecules called <i>N</i>-myristoyltransferase and calcineurin in barn air and <i>E. coli</i> LPS induced lung inflammation models. Following one day barn exposure, increased protein expression but not activity of <i>N</i>-myristoyltransferase and calcineurin was shown. However, there is a need to identify the specific role of these two molecules in barn air induced lung inflammation. To conclude, animal models of barn exposure are useful tools to understand mechanisms of lung inflammation and AHR. However, there is still a need to examine endotoxin-independent nature of AHR and roles of other molecules of the innate immune system in regulating barn air induced effects.

Occupational exposure to endotoxin-rich swine barn air induces respiratory diseases and loss of lung function. Barn exposure induces recruitment of pulmonary intravascular monocytes/macrophages (PIMMs) and subsequent increased host sensitivity to <i>Escherichia coli</i> LPS challenge. Therefore, to further clarify the biology of PIMMs we examined the role of recruited PIMMs in a rat <i>Escherichia coli</i>-induced lung inflammation model. Following sepsis, lung inflammation was induced with recruitment of PIMMs and subsequently, <i>Escherichia coli</i> LPS challenge exacerbated the lung inflammation with localization of multiple inflammatory cytokines in PIMMs to suggest their possible involvement in modulating lung inflammation in this model.<p> In order to delineate mechanisms of barn air induced lung dysfunction, a rat model of occupational exposure was characterized to show that one and five exposures to the barn environment induced acute lung inflammation and increased airway hyperresponsiveness (AHR). Following 20 exposures, AHR was dampened to indicate adaptive responses. Barn air contains high levels of endotoxin which led us to investigate its role in lung inflammation and AHR. Exposure of mice with either a functional TLR4 (WT) or non-functional TLR4 (mutants) to barn air revealed dependence of lung inflammation but not AHR on a functional TLR4.<p> I investigated whether exposure to barn air alters host responses to a subsequent microbial challenge. Following one day barn exposure and <i>Escherichia coli</i> LPS challenge, lung inflammation was exacerbated with increased granulocytes and IL-1β levels compared to one day barn exposed rats without <i>Escherichia coli</i> LPS challenge. However, increased granulocytes and IL-1β levels in barn exposed and <i>Escherichia coli</i> LPS challenged rats were not different from control rats treated with <i>Escherichia coli</i> LPS indicating a lack of priming effect of barn exposure. However, above results are suggestive of an underlying risk of increased lung inflammation following secondary microbial infection in naïve barn workers.<p> Lastly, I investigated the expression and activity of novel signalling molecules called <i>N</i>-myristoyltransferase and calcineurin in barn air and <i>E. coli</i> LPS induced lung inflammation models. Following one day barn exposure, increased protein expression but not activity of <i>N</i>-myristoyltransferase and calcineurin was shown. However, there is a need to identify the specific role of these two molecules in barn air induced lung inflammation. To conclude, animal models of barn exposure are useful tools to understand mechanisms of lung inflammation and AHR. However, there is still a need to examine endotoxin-independent nature of AHR and roles of other molecules of the innate immune system in regulating barn air induced effects.

Ce travail de thèse reposait sur l’hypothèse que la variabilité génétique des protéines « cibles » des médicaments immunosuppresseurs de la famille des inhibiteurs de la calcineurine (ICN ; ciclosporine et tacrolimus) pourrait expliquer une partie de la variabilité observée dans leur efficacité et toxicité. Une revue de la littérature nous a permis de lister un panel de variants génétiques au sein de la voie de la calcineurine, considérés comme étant de bons candidats pour des études en transplantation. Ces variants n’ont pas été associés au risque de rejet aigu ou d’infection grave dans une étude incluant 381 patients transplantés rénaux suivis durant un an après la transplantation. La variabilité pharmacodynamique des ICN a ensuite été explorée au travers des régulations épigénétiques. Une analyse de la méthylation de l’ADN après exposition médicamenteuse a été menée sur deux modèles. Premièrement, la lignée cellulaire JURKAT a été utilisée pour développer la méthode d’immunoprécipitation de l’ADN méthylé (MeDIP). Chez des souris traitées par ciclosporine et tacrolimus durant 3 mois, nous avons ensuite isolé les cellules cibles des médicaments, les lymphocytes T CD4 puis, après immunoprécipitation de l’ADN méthylé et analyse par séquençage pangénomique haut débit (MeDIP-seq, séquençeur Ion Proton), nous avons recherché les régions du génome présentant des différences de méthylation induites par le traitement. L’analyse différentielle bio-informatique a été menée à l’aide des outils SAMtools (Li et col., 2009), BEDtools (Quinlan and Hall, 2010), MACS2 (Zhang et col., 2008) et Diffbind (Stark and Brown, 2011 - Bioconductor). Sur l’ensemble du génome, nous n’avons identifié que 24 régions présentant un niveau de méthylation modifié par l’exposition au tacrolimus. Le promoteur du gène Calm2, codant pour l’isoforme 2 de la calmoduline, semble être davantage méthylé chez les souris traitées. Ces résultats préliminaires semblent prometteurs pour la découverte de biomarqueurs épigénétiques de la réponse thérapeutique aux immunosuppresseurs. / Inter-individual genetic variation might account for diverse efficacy and toxicity of calcineurin inhibitors (cyclosporin and tacrolimus). In particular, some variants located within genes coding for proteins of the calcineurin pathway can explain part of this variability. In this manuscript, a panel of candidate genes was selected based on bibliographic review and tested in a pharmacogenetics study encompassing 381 renal transplants followed for one year after surgery. None of these candidates was associated with the acute rejection or serious infection risks. Furthermore, the pharmacodynamic variability of these drugs was also investigated, exploring the use of epigenomics profiling as proximal readout of the calcineurin inhibition treatment. In particular, we investigated the impact of drug exposure on DNA methylation in two experimental models. Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq, Ion Proton technology) was deployed in JURKAT cell line, used as in vitro model, and in CD4 T lymphocytes isolated from mice treated with either cyclosporin or tacrolimus for three months. After sequencing, the differentiated methylated regions caused by drug exposure were analyzed. Bioinformatics analyses were performed using SAMtools (Li et al., 2009), BEDtools (Quinlan and Hall, 2010), MACS2 (Zhang et al., 2008) and Diffbind (Stark and Brown, 2011 - Bioconductor). Overall, the genome-wide analysis revealed only 24 regions with a differentiated enrichment in DNA methylation after three month-tacrolimus treatment, indicating a targeted effect of these treatments on a subset of key genes. Of note, CALM2 promoter, coding for the calmodulin isoform 2 protein, showed significant hypermethylation in tacrolimus-treated mice. These preliminary results corroborate the interest in using DNA methylation as promising approach to identify candidate biomarkers for therapeutic drug monitoring in calcineurin inhibitor treatments.