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.

For the past few decades, the popularity of graphene oxide (GO) nanomaterials (NMs) has increased exceedingly due to their biomedical applications in drug delivery of anti-cancer drugs. Their unique physicochemical properties such as high surface area and good surface chemistry with unbound surface functional groups (e.g. hydroxyl - OH, carboxyl /ketone C=O, epoxy/alkoxy C-O, aromatic group C=C, etc) which enable covalent bonding with organic molecules (e.g. RNA, DNA) make GO NMs as excellent candidates in drug delivery nanocarriers. Despite the overwhelming biomedical applications, there are concerns about their genotoxicity on human DNA. Published genotoxicity studies on GO NMs were performed using non-commercial GO with 2-3 layers of GO sheets, synthesized in various laboratories with the potential for inter-laboratory variabilities. However, what has not been studied before is the effects of the commercial GO (15-20 sheets; 4-10% edge-oxidized; 1 mg/mL) in vitro on DNA damage in human whole blood and peripheral blood lymphocytes (PBL) from real-life patients diagnosed with chronic pulmonary diseases [asthma, chronic obstructive pulmonary disease (COPD), and lung cancer], and genotoxic endpoints compared with those from healthy control individuals to determine whether there are any differences in GO sensitivity. Thus, in the present study, we had characterized GO NMs using Zetasizer Nano for Dynamic Light Scattering (DLS) and zeta potential (ZP) in the aqueous solution, and electron microscopy using the Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) in the dry state, respectively. Cytotoxicity studies were conducted on human PBL from healthy individuals and patients (asthma, COPD, and lung cancer) using the Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Neutral Red Uptake (NRU) assays, respectively. The genotoxicity (DNA damage) and cytogenetic effects (chromosome aberration parameters) induced by GO NMs on human whole blood from healthy individuals and patients were studied using the Alkaline Comet Assay and Cytokinesis-blocked Micronucleus (CBMN) assay, respectively. Our results showed concentration-dependent increases in cytotoxicity, genotoxicity, and chromosome aberrations, with blood samples from COPD and lung cancer patients being more sensitive to DNA damage insults compared with asthma patients and healthy control individuals. Furthermore, the relative gene and protein expressions of TP53, CDKN1A/p21, and BCL-2 relative to GAPDH on human PBL were studied using the Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and Western Blot techniques, respectively. Our results have shown altered gene and protein expression levels. Specifically, GO-induced cytotoxicity, genotoxicity, and micronuclei aberrations were associated with TP53 upregulation - a biomarker of DNA damage - in both patients and healthy individuals. These effects show that GO NMs have promising roles in drug delivery applications when formulated to deliver drug payload to COPD and cancer cells. However, the fact that cytotoxicity, genotoxicity, chromosome instability, and gene/protein expressions - biomarkers of cancer risk - were observed in healthy individuals are of concern to public health, especially in occupational exposures at micro levels at the workplace.

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.

No description available.

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.

Ο S. epidermidis αποτελεί κύριο μέλος της χλωρίδας του δέρματος και των βλεννογόνων του ανθρώπου, ενώ συνιστά ένα από τα συχνότερα παθογόνα που προκαλούν νοσοκομειακές λοιμώξεις, ιδιαίτερα σε ανοσοκατασταλμένους ή ασθενείς φέροντες προσθετικά υλικά. Κύριος λοιμογόνος παράγοντας του S. epidermidis είναι ο σχηματισμός βιομεμβράνης. Διάφοροι πολυσακχαρίτες έχουν απομονωθεί από την εξωκυττάρια ουσία του S. epidermidis και έχουν συσχετισθεί με το σχηματισμό βιομεμβράνης καθώς και με την παθογόνο δράση τους. Ο πλέον μελετημένος και εδραιωμένος είναι ο ΡΙΑ, ενώ οι άλλοι πολυσακχαρίτες (PS/A ή PNSG ή PNAG και το SSA) απεδείχθησαν τελικώς ότι είναι ταυτόσημοι ή χημικώς ανάλογοι του ΡΙΑ. Ο ΡΙΑ είναι μία ομογλυκάνη αποτελούμενη από μονάδες Ν-ακετυλογλυκοζαμίνης συνδεδεμένες με β-1,6-γλυκοζιτικό δεσμό και η σύνθεσή του ελέγχεται από ένζυμα που κωδικοποιούνται από τον icaADBC γενετικό τόπο. Η εξωκυττάρια ουσία του S. epidermidis περιέχει επίσης έναν πολυσακχαρίτη, τον 20-kDaPS, ο οποίος απομονώθηκε από ερευνητές του Πανεπιστημίου Πατρών και αποτελείται κυρίως από γλυκόζη, Ν-ακετυλογλυκοζαμίνη, και είναι μερικώς θειωμένος. Ο 20-kDaPS αντιορός αναστέλλει την προσκόλληση του S. epidermidis στα ενδοθηλιακά κύτταρα και την εμφάνιση βακτηριακής κερατίτιδας σε κουνέλια. Με βάση αναλύσεις κλινικών στελεχών, ο 20-kDaPS εκφράζεται σε μεγάλο ποσοστό στελεχών S. epidermidis, ενώ δεν ανευρέθηκε σε στελέχη άλλων πηκτάση αρνητικών σταφυλοκόκκων. Η παρεμβολή αλληλουχιών εισδοχής σε διάφορα σημεία του icaADBC οπερονίου, το οποίο ελέγχει τη σύνθεση του ΡΙΑ, δεν παρεμβάλλεται στην έκφραση του 20-kDaPS. Η κατεργασία βακτηριακών κυττάρων S. epidermidis με το ένζυμο dispersin B, το οποίο διασπά ειδικά τον β-1,6-γλυκοζιτικό δεσμό που συνθέτει το πολυμερές του ΡΙΑ, και με μετα-περιοδικό νάτριο που διασπά το δεσμό μεταξύ των ατόμων άνθρακα C-3 και C-4 των μονομερών Ν-ακετυλογλυκοζαμίνης, οδηγεί σε διάσπαση της βιομεμβράνης, χωρίς όμως να διαφοροποιείται αντιγονικά ο 20-kDaPS. Τα κλάσματα, μετά την έκλουση της εξωκυττάριας ουσίας του S. epidermidis από στήλη Q-Sepharose, που εμφανίζουν τη μεγαλύτερη ανοσοδραστικότητα για τον ΡΙΑ, στερούνται πλήρως 20-kDaPS. Η προεπώαση κλινικού στελέχους που δεν εκφράζει τον 20-kDaPS με τον πολυσακχαρίτη αναστέλλει την ενδοκυττάρωση, ενώ η προεπώαση του πρότυπου στελέχους ATCC35983 που συνθέτει τον 20-kDaPS με ειδικό αντιορό ενισχύει την ενδοκυττάρωση από τα ανθρώπινα μακροφάγα. Κατά συνέπεια, ο icaADBC γενετικός τόπος δεν εμπλέκεται στη σύνθεση του 20-kDaPS. Οι ανοσοχημικές και χρωματογραφικές ιδιότητες του PIA και του 20-kDaPS είναι διακριτές. Ο 20-kDaPS πιθανό να επιδεικνύει αντιφαγοκυτταρική δράση, ενώ τα ειδικά αντισώματα έχουν δράση οψωνίνης. Η οργάνωση των βακτηρίων εντός βιομεμβράνης προστατεύει από τις αντιμικροβιακές ουσίες και το σύστημα ανοσίας. Τα βακτήρια εντός βιομεμβράνης περιέχουν στην εξωκυττάρια ουσία τους μεγαλύτερα ποσά ΡΙΑ από τα ελεύθερα αναπτυσσόμενα, πλαγκτονικά κύτταρα. Τα βακτήρια εντός βιομεμβράνης επιδεικνύουν μεγαλύτερη ικανότητα για προσκόλληση και επιβίωση εντός των ανθρώπινων μακροφάγων από τα πλαγκτονικά κύτταρα. Τα βακτήρια εντός βιομεμβράνης επάγουν την παραγωγή μικρότερων ποσών φλεγμονωδών κυτταροκινών, όπως ο TNFα, καθώς και Th1 κυτταροκινών, όπως οι IL-12p40, IL-12p70 and IFN-γ, ενώ ενισχύουν τις IL-8, GM-CSF και IL-13. Οι συγκεκριμένες παρατηρήσεις αφορούν ζωντανά βακτηριακά κύτταρα καθώς και βακτηριακά κύτταρα μονιμοποιημένα με φορμαλδεΰδη. Τα ανωτέρω δεδομένα συνάδουν με την ήπια συμπτωματολογία των σχετιζόμενων με βιομεμβράνη λοιμώξεων S. epidermidis και τη διαφυγή της επιτήρησης του ανοσολογικού συστήματος. Συμπερασματικά, ο 20-kDaPS αποτελεί κύριο συστατικό της εξωκυττάριας ουσίας του S. epidermidis με αντιφαγοκυτταρικές και ανοσορρυθμιστικές ιδιότητες. Οι ανοσοχημικές και χρωματογραφικές ιδιότητες του 20-kDaPS είναι πλήρως διακριτές του ΡΙΑ, του κύριου πολυσακχαρίτη που σχετίζεται με το σχηματισμό βιομεμβράνης, ενώ ο γενετικός τόπος icaADBC δε ρυθμίζει τη σύνθεση του 20-kDaPS. Η οργάνωση των βακτηρίων εντός βιομεβράνη εξασφαλίζει αντίσταση στην ενδοκυττάρια θανάτωσή τους από τα μακροφάγα και καταστολή της ανοσιακής απόκρισης. / The skin commensal and opportunistic pathogen Staphylococcus epidermidis is a leading cause of hospital-acquired and biomaterial-associated infections. The polysaccharide intercellular adhesin (PIA), a homoglycan composed of β-1,6-linked N-acetylglucosamine residues, synthesized by enzymes encoded by the icaADBC operon is a major functional factor in biofilm accumulation, promoting virulence in experimental biomaterial-associated S. epidermidis infections. Extracellular mucous layer extracts of S. epidermidis contain another major polysaccharide, referred to as 20-kDa polysaccharide (20-kDaPS), composed mainly out of glucose, N-acetylglucosamine, and being partially sulfated. 20-kDaPS antiserum prevents adhesion of S. epidermidis on endothelial cells and development of experimental keratitis in rabbits. Here we provide experimental evidence that 20-kDaPS and PIA represent distinct molecules and that 20-kDaPS is implicated in endocytosis of S. epidermidis bacterial cells by human monocyte-derived macrophages. Analysis of 75 clinical coagulase-negative staphylococci from blood-cultures and central venous catheter tips indicated that 20-kDaPS is expressed exclusively in S. epidermidis but not in other coagulase-negative staphylococcal species. Tn917-insertion in various locations in icaADBC in mutants M10, M22, M23, and M24 of S. epidermidis 1457 are abolished for PIA synthesis, while 20-kDaPS expression appears unaltered as compared to wild-type strains using specific anti-PIA and anti-20-kDaPS antisera. While periodate oxidation and dispersin B treatments abolish immuno-reactivity and intercellular adhesive properties of PIA, no abrogative activity is exerted towards 20-kDaPS immunochemical reactivity following these treatments. PIA polysaccharide I-containing fractions eluted from Q-Sepharose were devoid of detectable 20-kDaPS using specific ELISA. Preincubation of non-20-kDaPS-producing clinical strains with increasing amounts of 20-kDaPS inhibits endocytosis by human macrophages, whereas, preincubation of 20-kDaPS-producing strain ATCC35983 with 20-kDaPS antiserum enhances bacterial endocytosis by human macrophages. In conclusion, icaADBC is not involved in 20-kDaPS synthesis, while the chemical and chromatographic properties of PIA and 20-kDaPS are distinct. 20-kDaPS exhibits anti-phagocytic properties, whereas, 20-kDaPS antiserum may have a beneficial effect on combating infection by 20-kDaPS-producing S. epidermidis. As stated above, biofilm formation is a major virulence factor of S. epidermidis. Biofilm protects bacterial cells from antimicrobial agents and components of the immune system. Interactions of peripheral blood mononuclear cells and monocyte derived macrophages with planktonic or biofilm phase S. epidermidis cells were also studied. Biofilm phase bacteria exhibited higher attachment, as well as, a ten fold higher intracellular survival in monocyte-derived macrophages than their planktonic counterparts. Stimulation of peripheral blood mononuclear cells and monocyte derived macrophages was performed with live or formalin-fixed bacterial cells. Supernatant concentration of selected cytokines was measured by Luminex® xMAP™ technology at different time points. As compared to planktonic phase, biofilm phase bacteria elicited lower amounts of proinflammatory cytokines and Th1 response cytokines, such as TNFα, IL-12p40, IL-12p70 and IFN-γ, whereas, they enhanced production of IL-8, GM-CSF and IL-13. This phenomenon was independent of formalin pretreatment. Taken together, these results may contribute to interpretation of observed silent course of biofilm associated infections. In conclusion, 20-kDaPS represents a major component of S. epidermidis extracellular matrix and data show that 20-kDaPS posseses antiphagocytic and immunomodulatory properties. 20-kDaPS and PIA are immunochemically and chromatographically discrete molecules, whereas icaADBC locus is not involved in 20-kDaPS synthesis. Biofilm mode of growth ensures higher resistance rates to intracellular killing and down regulation of immune responses.