O enfisema pulmonar define-se como a destruição das paredes alveolares e consequente dispneia progressiva. Este trabalho objetivou a adequação de um modelo de transplante celular in vivo de BMMC em camundongos com enfisema pulmonar. O enfisema foi induzido por instilação nasal de elastase (4 UI por animal). O diâmetro alveolar médio para os grupos não tratados e tratados com elastase apresentou diferença estatisticamente significativa, e mudanças no padrão de expressão de metaloproteinases envolvidas no processo inflamatório foram detectadas, indicando que a instilação de uma dose de elastase promove lesão semelhante ao enfisema pulmonar. Infundiu-se 0,4ml de BMMC (7x106 céls./ml) nestes animais. No grupo tratado com células, detectou-se mudanças morfométricas e no padrão de expressão de metaloproteinases, indicando melhora na evolução da lesão pulmonar 21 dias após a infusão. Foram ainda avaliadas duas e três doses do pool de BMMC, porém os resultados das análises mostraram que não há diferenças entre estre grupos e os grupos controle. / Pulmonary emphysema is defined as the destruction of the alveolar walls and consequent progressive dyspnea. This study aimed the adequacy of a model of BMMC transplantation in vivo in mice with pulmonary emphysema. Emphysema was induced by nasal instillation of elastase (4 IU per animal). The mean linear intercept for the groups untreated and treated with elastase showed a statistically significant difference, and changes in the pattern of expression of metalloproteinases involved in inflammation were detected, indicating that the instillation of a dose of elastase promotes lung damage similar to emphysema. 0.4 ml of BMMC (7x106 céls. / ml) was infused in these animals. In the group treated with cells there were detected and morphometric changes in the pattern of expression of metalloproteinases, indicating an improvement in the evolution of lung injury 21 days after infusion. Were also evaluated two and three doses of the pool BMMC, but the results of the analysis showed no differences between experimental and the control groups.

PhD Aims: The aim of our study was to evaluate an efficacy and safety of intra-arterial injection of bone marrow mononuclear cells (BMMCs) in patients with chronic critical limb ischemia (CLI) Methods.In average 400ml bone marrow blood was harvested from posterior iliac crests in 24 CLI patients. BMMCs were obtained from the blood by standard procedure used for bone marrow transplantation. After digital subtraction angiography was performed in each patient, BMMCs were injected into arteries of 28 limbs. Primary outcome was the efficacy of BMMCs injection measured as a successfull healing of limb defects, a change of Fontain ischemia grade and a rate of high limb amputations. Secondary outcomes were a safety of the BMMCs injections, changes in angiographic findings after BMMCs injections and changes in quality of life (questionnaire SF-36). Results: After one year follow-up all patients were alive and 2 patients have undergone high limb amputation. Out of 14 limb defects, eleven have been healed completely and the average Fontain ischemia grade has changed from baseline value of 3.5 to 2.0 after one year (P<0.0001). Angiographic findings have improved in all examined segments of limb vessels. One year after the procedure patients have reported significant improvement. Conclusion: The intra-arterial...

Cardiovascular disease is the main cause of death in the United States. Although it is recognized that moderate intensity long-term exercise can decrease the chances of dying from cardiovascular disease by favorably modifying risk factors such as hypertension, obesity, hyperlipidemia, and insulin resistance, physical activity also enhances longevity by mechanisms independent of these risk factors. This review briefly summarizes what is known about the inflammatory nature of atherosclerosis and how long-term aerobic exercise can reduce the atherogenic activity of endothelial cells, blood mononuclear cells, and adipose tissue.

The local release of complexed siRNA from biomaterials opens precisely targeted therapeutic options. In this study, complexed siRNA was loaded to gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA). We aggregated these siRNA-loaded cGM with human mesenchymal stem cells (hMSC) to microtissues and stimulated them with osteogenic supplements. An efficient knockdown of chordin, a BMP-2 antagonist, caused a remarkably increased alkaline phosphatase (ALP) activity in the microtissues. cGM, as a component of microtissues, mineralized in a differentiation medium within 8–9 days, both in the presence and in the absence of cells. In order to investigate the effects of our pre-differentiated and chordin-silenced microtissues on bone homeostasis, we simulated in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). We found enhanced ALP activity and osteoprotegerin (OPG) secretion in the model system compared to control microtissues. Our results suggest osteoanabolic effects of pre-differentiated and chordin-silenced microtissues.

Human Immunodeficiency Virus (HIV) is a virus that is transmitted through certain bodily fluids and compromises the immune system of its host. Despite the emergence of antiretroviral therapy (ART) converting human immunodeficiency virus type 1 (HIV-1) infection from a fatal disease to a chronic condition, there is still no cure. ART frequently reestablishes peripheral CD4+ T cell counts, but persistent immune dysfunction and inflammation strongly correlate with increased risks of attaining non-AIDS morbidity and mortality. Elimination of this reservoir may occur by the proposed mechanism of combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T cells (Meås et al., 2020). Here, our study investigates Toll-like receptor 7/8 (TLR 7/8) superagonists that may act as potent, effective latency reversal agents (LRAs). Whether this will prove to be the case needs to be further studied, and potential adverse toxicities must be identified. Whether comparable results will be observed in peripheral blood mononuclear cells (PBMCs) infected with HIV-1 as in our study using PBMCs infected with simian immunodeficiency virus (SIV) remains to be tested. Our results provide further hope for a potential cure for HIV-infected individuals.

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2007. / SUPERFICIAL digital flexor tendon injury has a serious negative impact on the competitive horse industry. Injured horses require up to a year of rest for recovery and likelihood of re-injury upon return to normal activity is as high as 80 %. Tendon healing requires (a) production of collagen by fibroblasts, to provide tensile strength and elasticity to the tendon, (b) minimisation of restrictive fibrosis, which compromises tendon gliding function and (c) minimisation of peritendinous adhesions. We review conventional treatments for tendon healing before exploring stem cell application as a therapeutic alternative. We promote the use of hematopoietic and mesenchymal stem cells derived from adult peripheral blood - as opposed to bone marrow-derived stem cells or embryonic stem cell sources - and review published research output in this regard. In conclusion, we outline our research objectives and present and discuss our results in the chapters that follow. Mononuclear cells - consisting of hematopoietic stem cells, mesenchymal stem cells and leucocytes – were isolated from the peripheral blood of sheep and horses through red blood cell lysis and blood plasma extraction. Cell counts and propidium iodide dye exclusion viability tests were conducted on the cell pellets. Sheep sub samples were tested for CD45 expression and horse sub samples for CD4 and CD11a/18 cell surface markers by flow cytometry for characterisation purposes. In both cases, separate sub samples were incubated with matched immunoglobulin (IgG) isotypes, conjugated to fluorescein isothiocyanate (FITC), to serve as controls. For the culture of mononuclear cells, 4.5 x 106 cells were selected for autologous sheep injections, 3 x 106 CD45- cells for allogeneic sheep injections (the latter excluding leucocytes that may induce an immune response) and 72 x 106 cells for horse injections. These cells were incubated with bromo-deoxyuridine (BrdU), cultured and subsets were extracted for a second round of cell counts and viability tests before being resuspended in blood plasma. For the horse samples an additional 1 x 106 mononuclear cells were incubated until reaching 60 % confluence and tested for myogenic differentiation. Low cell mortality and lack of fluorescence from IgG-FITC controls reflected effective protocols and a lack of false positive results. The fact that the equine cell population differentiated into myotubes verified the presence of mesenchymal stem cells in injections. We tested whether surgical incisions or collagenase injections best mimicked naturally occurring tendon injuries and compiled macroscopic and microscopic descriptions of tendon injury sites at seven weeks post-injury. The superficial digital flexor tendons of 27 sheep received an incision, a collagenase injection or a saline control injection. After one week a number of sheep were sacrificed while the remainder received further saline treatment and were sacrificed after another seven weeks. Tendons were examined through clinical observations, image analysis of maximum tendon diameter, mechanical testing and histological sectioning of affected tissues. Collagenase-induced injury resembled tendonitis more closely than surgically-induced injury. Collagenase-injured tendons (a) induced lengthier lameness in affected limbs, (b) were more swollen and difficult to palpate, (c) assumed the bow appearance characteristic of natural injury, (d) experienced extensive haemorrhage due to collagen lysis, (e) had decreased elasticity and capacity to carry loads and stress, (f) displayed decreased stiffness due to collagen fibre disruption and (g) developed severe inflammation. After seven weeks injured tendons displayed increased vascularisation in the areas of haemorrhage and in the adjacent collagen matrix. High inflammation rates and low collagen levels however still persisted. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of 27 sheep. After one week these tendons received treatment with a control saline solution, autologous peripheral blood mononuclear cells (MNCs) or allogeneic peripheral blood CD45- MNCs. Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analyses of maximum tendon diameter, mechanical tests and histological investigations. Tendons treated with MNCs displayed an improvement in echogenicity and fibre linearity, higher and more organised collagen levels, stronger mechanical properties and less swelling. Although these improvements were not always significant, they provided strong evidence to suggest marked healing benefits over a longer time period. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of four horses. After one week these tendons received treatment with either a control saline solution or autologous peripheral blood mononuclear cells (MNCs). Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analysis of maximum tendon diameter and histological investigations. Tendons treated with MNCs displayed significant improvements in fibre linearity in the direct vicinity of the lesion, as well as recovery rate thereof, and experienced less swelling when compared with their untreated counterparts. Healing trends suggested that, given a longer period of observation post-injury, more significant improvements may become apparent. Human adipose tissue is known be an easily accessible and high yielding source of multipotent mesenchymal stem cells. These stem cells could potentially be used for therapeutic advancement of tendon regeneration. Our first goal was to examine the in vitro myogenic differentiation potential of adipose-derived, adherent mononuclear cells (MNCs) from six adult sheep. The second goal was to characterise the population of cells isolated through various available ovine specific, non-mesenchymal stem cell surface markers, namely, CD1, CD31, CD34 and CD45. After incubation, only four of the six MNC cultures started to proliferate. These four cultures all exhibited high myogenic differentiation ability. The isolated cell populations did not express any of the non-mesenchymal stem cell specific cell surface markers. In conclusion, our data suggests that peripheral blood stem cells and adipose-derived stem cells are important candidate cell types for therapeutic application to improve tendon repair in horses and sheep. Sufficient time must be allowed following injury and prior to stem cell treatment (at least one month) and a controlled exercise program should be followed posttreatment. A larger sample size is required and at least six months of recovery before macroscopic and histological repair can be analysed more accurately and conclusively. Ultrasonography should be carried out on a continuous basis, as it is a non-invasive method of monitoring change over time.

A doação de plaquetas por aférese é um procedimento automatizado que permite a obtenção deste hemocomponente em grande quantidade e com ato grau de pureza; deste processo obtém-se um subproduto chamado Câmara de Leucorredução (CLR) que é descartado ao final da doação. São permitidas até 24 doações/ano; porém as possíveis consequências de doações frequentes para esses doadores são pouco investigadas. Assim, foram identificados e quantificados os leucócitos de doadores de plaquetas frequentes e de 1ª vez. Também foi avaliada a viabilidade do uso das células mononucleares da CLR para pesquisas. Observou-se mais células na CLR que no sangue e que a frequência das populações é similar. O estado de ativação e a capacidade funcional (proliferação e produção de citocinas) foram similares entre CLR e sangue, assim como a taxa de apoptose espontânea. Entre doadores frequentes e de primeira vez não houve diferença no número de leucócitos, sugerindo que doações recorrentes não alteraram as populações leucocitárias. / Plateletpheresis is an automatized procedure to obtain high purity platelet for transfusions. From this procedure its possible to obtain a byproduct: The Leukoreduction system chamber (LRSC), which is discarded at the end of donation process. This type of donation allows 24 donation/year, but the consequences of frequent donations are poorly investigated. Therefore, we identified and quantified leukocytes of frequent and first time platelet donor. Also, was evaluated the viability, for research, of mononuclear cells recovery from LRSC. The total number of mononuclear cells was higher in LRSC than in peripheral blood samples, but the frequencies were similar in all the samples. Activation state and functional capacity (measured by cell proliferation and cytokine production) were similar in both, blood and LRSC mononuclear cells, as well as spontaneous apoptosis. Among frequent (6 or more donations in 1 year) and first time donor, there was no difference in the leukocyte total number, suggesting that frequent donation do not modify these cells.

A esclerose múltipla (EM), doença neurodegenerativa do sistema nervoso central (SNC) com característica auto-imune e inflamatória, com eventos iniciais, bem como a evolução da EM. É uma doença heterogênea (três principais formas clínicas) e multifatoriais. A imunidade inata demonstrou recentemente ser um fator importante na EM e as variantes genéticas dos componentes do inflamassoma têm sido associadas a doenças autoimunes e neurodegenerativas, com isso hipotetizamos que o inflamassoma e suas citocinas IL-1Beta e IL-18, podem representar importantes contribuintes na patogênese da EM e eventualmente explicar, pelo menos em parte, a heterogeneidade observada em pacientes com EM. Fizemos uma análise multivariada que foi realizada com base na forma clínica (recorrente remitente/RR, primária progressivo /PP ou secundário progressiva /SP, índice de gravidade (EDSS) e índice de progressão (IP). Os monócitos do sangue periférico (PBMC) dos pacientes foram examinados para ativação do inflamassoma (Produção de IL-1Beta e IL-18, clivagem de caspase-1). Com os objetivos de avaliar a contribuição do inflamassoma na EM, em termos de (a) efeito genético sobre o desenvolvimento, gravidade e / ou prognóstico, e (b) ativação complexa de células de sangue periférico como uma forma de avaliar a inflamação sistêmica. Para isso, utilizamos variantes genéticas funcionais em componentes do inflamassoma, que foram analisadas em uma coorte de pacientes com EM, pelo uso de ensaios específicos de alelos e qPCR. A analise multivariada resultou em associação com a variante -511C / T IL1B ganho de função, sendo essa mais frequente em formas progressivas (especialmente SP) do que em RR. A variante de ganho de função NLRP3 Q705K resultou mais frequente em pacientes com EDSS > 3 do que em pacientes com EDSS < 3 e, consequentemente, esse SNP está associado a um IP mais elevado. A análise de PBMC mostrou que as células de indivíduos EM, são mais propensas a responder a um estímulo NLRP3 clássico (isto é, LPS) do que as dos doadores saudáveis. Em conjunto, esses achados indicaram que os pacientes com EM apresentam uma desregulação no inflamassoma NLRP3, podendo ser avaliada no sangue periférico facilitando um prognóstico, e que esse perfil pode ser secundário a um mecanismo genético pró-inflamassoma / The multiple sclerosis (MS), neurodegenerative disease of the central nervous system (CNS) with autoimmune and inflammatory characteristics, with initial events, as well as the evolution of MS, are heterogeneous (three main clinical forms) and multifactorial. Innate immunity has recently been shown to be an important factor in MS and the genetic variants of the components of inflammassoma have been associated with autoimmune and neurodegenerative diseases, thereby hypothesizing that the inflammassoma and its IL-1Beta and IL-18 cytokines may represent important contributors in the pathogenesis of MS and possibly explain, at least in part, the heterogeneity observed in MS patients. We performed a multivariate analysis that was performed based on clinical form (recurrent recurrent / RR, progressive primary / PP or progressive secondary / SP, EDSS and progression index.) Peripheral blood mononuclear cells (PBMC) of patients were examined for inflammatory activation (IL-1Beta and IL-18 production, caspase-1 cleavage). With the objectives of evaluating the contribution of inflammassoma in MS in terms of (a) genetic effect on development, severity and / or prognosis, and (b) complex activation of peripheral blood cells as a way of assessing systemic inflammation. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. Multivariate analysis resulted in association with the -511C / T IL1B function gain, which is more frequent in progressive forms (especially SP) than in RR. The gain variant of NLRP3 Q705K function was more frequent in patients with EDSS > 3 than in patients with EDSS < 3 and, consequently, this SNP is associated with a higher PI. PBMC analysis showed that cells from MS individuals are more likely to respond to a classical NLRP3 (ie LPS) stimulus than healthy donors. Taken together, these findings indicated that patients with MS have a dysregulation in the NLRP3 inflammassoma and can be evaluated in the peripheral blood facilitating a prognosis and that this profile may be secondary to a pro-inflammatory genetic mechanism

A esclerose múltipla (EM), doença neurodegenerativa do sistema nervoso central (SNC) com característica auto-imune e inflamatória, com eventos iniciais, bem como a evolução da EM. É uma doença heterogênea (três principais formas clínicas) e multifatoriais. A imunidade inata demonstrou recentemente ser um fator importante na EM e as variantes genéticas dos componentes do inflamassoma têm sido associadas a doenças autoimunes e neurodegenerativas, com isso hipotetizamos que o inflamassoma e suas citocinas IL-1Beta e IL-18, podem representar importantes contribuintes na patogênese da EM e eventualmente explicar, pelo menos em parte, a heterogeneidade observada em pacientes com EM. Fizemos uma análise multivariada que foi realizada com base na forma clínica (recorrente remitente/RR, primária progressivo /PP ou secundário progressiva /SP, índice de gravidade (EDSS) e índice de progressão (IP). Os monócitos do sangue periférico (PBMC) dos pacientes foram examinados para ativação do inflamassoma (Produção de IL-1Beta e IL-18, clivagem de caspase-1). Com os objetivos de avaliar a contribuição do inflamassoma na EM, em termos de (a) efeito genético sobre o desenvolvimento, gravidade e / ou prognóstico, e (b) ativação complexa de células de sangue periférico como uma forma de avaliar a inflamação sistêmica. Para isso, utilizamos variantes genéticas funcionais em componentes do inflamassoma, que foram analisadas em uma coorte de pacientes com EM, pelo uso de ensaios específicos de alelos e qPCR. A analise multivariada resultou em associação com a variante -511C / T IL1B ganho de função, sendo essa mais frequente em formas progressivas (especialmente SP) do que em RR. A variante de ganho de função NLRP3 Q705K resultou mais frequente em pacientes com EDSS > 3 do que em pacientes com EDSS < 3 e, consequentemente, esse SNP está associado a um IP mais elevado. A análise de PBMC mostrou que as células de indivíduos EM, são mais propensas a responder a um estímulo NLRP3 clássico (isto é, LPS) do que as dos doadores saudáveis. Em conjunto, esses achados indicaram que os pacientes com EM apresentam uma desregulação no inflamassoma NLRP3, podendo ser avaliada no sangue periférico facilitando um prognóstico, e que esse perfil pode ser secundário a um mecanismo genético pró-inflamassoma / The multiple sclerosis (MS), neurodegenerative disease of the central nervous system (CNS) with autoimmune and inflammatory characteristics, with initial events, as well as the evolution of MS, are heterogeneous (three main clinical forms) and multifactorial. Innate immunity has recently been shown to be an important factor in MS and the genetic variants of the components of inflammassoma have been associated with autoimmune and neurodegenerative diseases, thereby hypothesizing that the inflammassoma and its IL-1Beta and IL-18 cytokines may represent important contributors in the pathogenesis of MS and possibly explain, at least in part, the heterogeneity observed in MS patients. We performed a multivariate analysis that was performed based on clinical form (recurrent recurrent / RR, progressive primary / PP or progressive secondary / SP, EDSS and progression index.) Peripheral blood mononuclear cells (PBMC) of patients were examined for inflammatory activation (IL-1Beta and IL-18 production, caspase-1 cleavage). With the objectives of evaluating the contribution of inflammassoma in MS in terms of (a) genetic effect on development, severity and / or prognosis, and (b) complex activation of peripheral blood cells as a way of assessing systemic inflammation. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. For this, we used functional genetic variants in components of the inflammassoma, which were analyzed in a cohort of MS patients, through the use of specific allele and qPCR assays. Multivariate analysis resulted in association with the -511C / T IL1B function gain, which is more frequent in progressive forms (especially SP) than in RR. The gain variant of NLRP3 Q705K function was more frequent in patients with EDSS > 3 than in patients with EDSS < 3 and, consequently, this SNP is associated with a higher PI. PBMC analysis showed that cells from MS individuals are more likely to respond to a classical NLRP3 (ie LPS) stimulus than healthy donors. Taken together, these findings indicated that patients with MS have a dysregulation in the NLRP3 inflammassoma and can be evaluated in the peripheral blood facilitating a prognosis and that this profile may be secondary to a pro-inflammatory genetic mechanism

Les immunosuppresseurs sont des médicaments ayant démontré leur efficacité dans la prévention du rejet de transplantation d’organe solide. Néanmoins, ces médicaments présentent une importante variabilité de la réponse pharmacologique notamment liée à une variation de leur pharmacocinétique. Cette variabilité peut être à l’origine d’un défaut d’efficacité du traitement ou de sur-expositions entrainant une toxicité. Le suivi thérapeutique pharmacologique (STP) des concentrations sanguines d’immunosuppresseur permet de limiter ces risques de sur ou de sous-exposition en facilitant l’adaptation de posologie de ces traitements. En dépit de l'utilisation intensive du STP des concentrations d'immunosuppresseurs chez le transplanté d'organe, la fréquence du rejet aigu a peu diminué au cours de ces dernières années et reste élevée. Le rejet aigu cellulaire peut, en outre, survenir chez le patient alors même que ses concentrations sanguines sont en zone thérapeutique. C'est pourquoi la recherche d'améliorations de ce suivi thérapeutique ainsi que la recherche de nouveaux moyens de monitoring sont des axes pertinents d'investigation en pharmacologie des immunosuppresseurs. L’objectif de ce travail de doctorat a été de développer de nouveaux outils pharmacologiques de monitoring de l’effet de deux immunosuppresseurs, l’évérolimus et le tacrolimus, dans le but de participer à la maitrise de la variabilité pharmacologique de l’effet immunosuppresseur. Pour la première fois, la pharmacocinétique de l'évérolimus chez le transplanté cardiaque a été modélisée par une approche de population. La modélisation pharmacologique est un des axes actuels d’amélioration du STP des immunosuppresseurs permettant d’évaluer l’impact de covariables démographiques, biologiques et/ou génétiques sur la pharmacocinétique de ces médicaments. L’élaboration de ce modèle doit permettre une individualisation des posologies conduisant à la limitation de la variabilité pharmacocinétique lors d’un traitement par cette molécule.Au cours de ce travail, deux méthodes analytiques ont également été développées par LC-MS/MS pour le dosage intracellulaire de l’évérolimus et du tacrolimus. La mesure des concentrations des immunosuppresseurs à l’intérieur même de la cellule, c’est à dire au niveau de son site d’action, apparait comme une mesure plus pertinente que la simple mesure des concentrations dans le sang. Ces méthodes ont ensuite été évaluées sur des petites cohortes de patients transplantés cardiaques. La faisabilité de tels dosages a été démontrée et a permis la réalisation de la dernière partie présentée au cours de ce travail. En effet, une étude clinique a été réalisée chez le patient transplanté hépatique de novo en vue de rapprocher les concentrations sanguines et intracellulaires de tacrolimus et l'effet sur la protéine cible, la calcineurine. Pour la première fois des pharmacocinétiques intracellulaires complètes ont pu être obtenues permettant la description de la cinétique intracellulaire du tacrolimus. Cette étude a également permis de mettre en évidence et de décrire la relation dose - concentration sanguine - concentration intracellulaire - effet sur la protéine cible du tacrolimus chez le transplanté hépatique. Ces travaux ont permis de jeter les bases nécessaires à la réalisation d'essais cliniques permettant d'évaluer la pertinence d'un suivi longitudinal des concentrations intracellulaires et/ou de l'activité de la calcineurine dans la prévention de rejet de transplantation. Les outils développés au cours de ce travail de doctorat visent, d’une part, à mieux appréhender la variabilité de la réponse pharmacologique au cours d’un traitement immunosuppresseur et, d’autre part, à être des outils de compréhension des mécanismes pharmacocinétiques et cellulaires de ces traitements. L'utilisation de ces outils doit concourir à la diminution de la fréquence du rejet de greffe et à l'amélioration globale de la prise en charge du patient transplanté d'organe. / Immunosuppressive drugs have proven efficacy in the prevention of acute rejection of solid organ transplantation. However, these drugs exhibit substantial variability in pharmacological response due to such a variation in their pharmacokinetics. This variability may be the cause of underexposure with a lack of efficacy or over-exposure causing toxicity. Therapeutic drug monitoring (TDM) of immunosuppressant blood levels can limit the risk of over or underexposure facilitating dosage adjustment of these treatments. Despite the extensive use of TDM, the incidence of acute rejection has declined somewhat in recent years and remains high (in the order of 8-15%). Acute cellular rejection can further occur in patients even though blood levels are within the therapeutic range. That is why improvements in the therapeutic monitoring and new ways of monitoring are relevant lines of investigation in pharmacology.The objective of this phD work was to develop new pharmacological tools for monitoring the effect of two immunosuppressive drugs, everolimus and tacrolimus in order to control the pharmacological variability of the immunosuppressive effect .For the first time, the pharmacokinetics of everolimus in heart transplant was modeled by a population approach. Pharmacological modeling is one of the current areas of improvement of immunosuppressants TDM which allows evaluating the impact of demographic, biological and / or genetic on the pharmacokinetics of these drugs covariates. The development of this model must allow individualization of dosages leading to limit pharmacokinetic variability during treatment with this drug.During this work, two analytical methods were also developed by LC-MS/MS for assaying intracellular tacrolimus and everolimus concentrations. Measuring intracellular concentrations of immunosuppressive drugs, i.e. at its site of action, appears as a more relevant than measuring blood concentrations. These methods were then evaluated on small cohorts of heart transplant patients. The feasibility of such assays has been demonstrated and led to the completion of the last part presented in this work.Indeed, a clinical study was performed in de novo liver transplant patients to evaluate blood and intracellular concentrations of tacrolimus and their effect on the target protein, calcineurin. For the first time complete intracellular pharmacokinetics have been obtained for the description of the profile of the intracellular kinetics of tacrolimus. This study also highlights and describes the relationship dose - blood concentration - intracellular concentration - effect on the target protein of tacrolimus in liver transplant patients. This work might help conducting clinical trials to assess the relevance of a longitudinal follow-up of intracellular concentrations and / or activity of calcineurin in the prevention of transplant rejection.The tools developed in this PhD work aimed, firstly, to better understand the variability of the pharmacological response in immunosuppressive therapy and, secondly, to be tools for understanding the drug mechanisms inside of the cell. The use of these tools should contribute to the decrease in the frequency of graft rejection and the overall improvement in the management of organ transplant patients.