<p>The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (T_H) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. </p><p>To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65_495-503 peptide, a recombinant adenovirus coding for pp65, <i>in vitro</i> transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8⁺ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4⁺ T_H cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and T_H cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells <i>ex vivo</i> from virtually all stem cell donors for adoptive T cell transfer. </p><p>I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8⁺ T cells even when starting from undetectable frequencies (<0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8⁺ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer. </p>

Background: Stem cell therapy can facilitate cardiac repair after healed myocardial infarction but the optimal cell type remains uncertain. Aims: To investigate the pathophysiology of heart failure in a canine model of healed myocardial infarction and to compare the efficacy and the safety of autologous bone marrow mononuclear cell (BMNC) transfer and mesenchymal stem cell (MSC) transfer in this model. It was a blind, randomized and placebo control study. Methods: Eleven weeks after coronary ligation, 24 dogs received intramyocardial injections of BMNC, MSC or Placebo (n = 8 per groups). Echocardiography, conductance method, magnetic resonance imaging, serum neurohormones, holter monitoring, macromorphometry, histology and real time quantitative polymerase chain reaction were used to assess cardiac performance, safety and remodelling in healthy animals, before cell transplantation and up to 16 weeks’ follow-up. Results: The model was characterized by decreased left ventricular end-systolic elastance and ventricular-arterial uncoupling without alteration of compliance. Four months after BMNC transfer, the regional systolic function measured at echocardiographic showed a sustained improvement. This improvement was associated with an improved left ventricular end-systolic elastance and a decreased infarct size. Although the left ventricular ejection fraction stayed unchanged, the serum level of N-terminal B-type natriuretic propeptide level decreased. Mononuclear cell transfer was also associated with increased left ventricular relative wall area, increased vascular density, intramyocardial vascular remodelling and upregulation of angiogenic factors gene expression. Mesenchymal stem cell transfer only improved lately and moderately the regional systolic function, without improvement of cardiac contractility or decreased infarct size. Conclusions: In a canine model of chronic myocardial infarction, BMNC transfer is superior to MSC transfer in improvement of cardiac contractility and regional systolic function, and to reduce the infarct size and plasma N-terminal B-type natriuretic propeptide level. Functional improvement is associated with a favourable angiogenic environment and neovascularization.

The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (TH) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65495-503 peptide, a recombinant adenovirus coding for pp65, in vitro transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8+ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4+ TH cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and TH cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells ex vivo from virtually all stem cell donors for adoptive T cell transfer. I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8+ T cells even when starting from undetectable frequencies (&lt;0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8+ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer.

Cardiovascular diseases are the leading cause of death throughout the world and various estimates predict that heart disease will remain the number one killer in the world. Pharmacotherapies have not shown significant long term survival benefits to the patients, therefore alternate therapeutic strategies such as bioactive agent delivery and cell therapy based approaches are being investigated. One of the major causes of heart failure is the disease progression after an ischemic event and any successful therapy will be needed over the course of several days/weeks. Oxidative stress is greatly increased in the myocardium following infarction. This plays a significant role in cardiac disease progression and it has also been implicated in the failure of implanted cell therapy. Therefore, reducing oxidative stress in damaged tissue using antioxidants may have broad clinical implications for both the treatment of cardiac dysfunction and for cardiac regeneration applications. This dissertation work examines the effect of sustained delivery of endogenous antioxidant superoxide dismutase (SOD) to the rat myocardium following ischemia/reperfusion (IR) using polyketal polymers as drug carriers. The second major objective of this dissertation is to examine the effects of oxidative stress on cardiac progenitor cells - a promising endogenous adult stem cell in cardiac cell therapy applications

No description available.

The acute respiratory distress syndrome (ARDS) and its less severe form, acute lung injury (ALI), are among the leading causes of morbidity and mortality in critically ill patients. Commonly induced by conditions associated with severe pulmonary inflammation, ALI results in disruption of the lung alveolar-capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudate. Sepsis is another frequent and often fatal clinical condition for patients in the intensive care unit. It is characterized by a combination of infection and systemic inflammatory response syndrome (SIRS). Current effective treatment strategies for both ALI/ARDS and sepsis are lacking. We first examined the potential therapeutic role of mesenchymal stromal cells (MSCs) alone or together with the vasculoprotective factor, angiopoietin-1 (ANGPT1), for treatment of experimental ALI in mice. MSCs significantly reduced LPS (lipopolysaccharide)-induced pulmonary inflammation, as reflected by cell counts in bronchoalveolar lavage (BAL) fluid and pro-inflammatory cytokine levels in both BAL fluid and lung parenchymal homogenates. More importantly, administration of MSCs transfected with human ANGPT1 plasmid (MSCs-pANGPT1) completely reversed LPS-induced permeability in the lung (i.e., ALI). A follow-up study showed that MSCs remained effective in rescuing mice with LPS-induced ALI; however, the additional benefit from ANGPT1 was no longer observed. To further evaluate MSC-based therapy in a more clinically relevant model of acute injury, the cecal-ligation-and-puncture (CLP) model for sepsis was employed. Our results demonstrated that MSCs can reduce both systemic and pulmonary inflammation, as well as renal and liver dysfunction/injury, as reflected by plasma urea and bilirubin levels, in septic mice. Most notably, MSCs reduced sepsis-associated mortality from 45% to 24%. Our data demonstrate the feasibility and effectiveness of MSC- and MSC-based gene therapy for experimental ALI and sepsis, and provide the basis for the development of an innovative approach for the prevention and treatment of clinical ALI/ARDS and sepsis.

The acute respiratory distress syndrome (ARDS) and its less severe form, acute lung injury (ALI), are among the leading causes of morbidity and mortality in critically ill patients. Commonly induced by conditions associated with severe pulmonary inflammation, ALI results in disruption of the lung alveolar-capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudate. Sepsis is another frequent and often fatal clinical condition for patients in the intensive care unit. It is characterized by a combination of infection and systemic inflammatory response syndrome (SIRS). Current effective treatment strategies for both ALI/ARDS and sepsis are lacking. We first examined the potential therapeutic role of mesenchymal stromal cells (MSCs) alone or together with the vasculoprotective factor, angiopoietin-1 (ANGPT1), for treatment of experimental ALI in mice. MSCs significantly reduced LPS (lipopolysaccharide)-induced pulmonary inflammation, as reflected by cell counts in bronchoalveolar lavage (BAL) fluid and pro-inflammatory cytokine levels in both BAL fluid and lung parenchymal homogenates. More importantly, administration of MSCs transfected with human ANGPT1 plasmid (MSCs-pANGPT1) completely reversed LPS-induced permeability in the lung (i.e., ALI). A follow-up study showed that MSCs remained effective in rescuing mice with LPS-induced ALI; however, the additional benefit from ANGPT1 was no longer observed. To further evaluate MSC-based therapy in a more clinically relevant model of acute injury, the cecal-ligation-and-puncture (CLP) model for sepsis was employed. Our results demonstrated that MSCs can reduce both systemic and pulmonary inflammation, as well as renal and liver dysfunction/injury, as reflected by plasma urea and bilirubin levels, in septic mice. Most notably, MSCs reduced sepsis-associated mortality from 45% to 24%. Our data demonstrate the feasibility and effectiveness of MSC- and MSC-based gene therapy for experimental ALI and sepsis, and provide the basis for the development of an innovative approach for the prevention and treatment of clinical ALI/ARDS and sepsis.

Submitted by DANIELE LOPES PEREIRA null (danielelopesp@hotmail.com) on 2016-05-11T22:02:11Z No. of bitstreams: 1 dissertação maio - 2016.doc: 36656128 bytes, checksum: 544a8bb80add41fb7e4e0faa6dc3b70c (MD5) / Rejected by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: A versão final da dissertação/tese deve ser submetida no formato PDF (Portable Document Format). O arquivo PDF não deve estar protegido e a dissertação/tese deve estar em um único arquivo, inclusive os apêndices e anexos, se houver. E também, o arquivo submetido está sem a folha de aprovações providenciada pela Pós-graduação. Lembramos que a versão submetida por você é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração em seu conteúdo após a aprovação. Corrija estas informações e realize uma nova submissão contendo o arquivo correto. Agradecemos a compreensão. on 2016-05-13T16:38:51Z (GMT) / Submitted by DANIELE LOPES PEREIRA null (danielelopesp@hotmail.com) on 2016-08-12T21:37:08Z No. of bitstreams: 1 Terapia celular em fibrose pulmonar.pdf: 14681718 bytes, checksum: ddd650e002e44e6ba3817e66564c1581 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-15T19:52:38Z (GMT) No. of bitstreams: 1 pereira_dl_me_assis.pdf: 14681718 bytes, checksum: ddd650e002e44e6ba3817e66564c1581 (MD5) / Made available in DSpace on 2016-08-15T19:52:38Z (GMT). No. of bitstreams: 1 pereira_dl_me_assis.pdf: 14681718 bytes, checksum: ddd650e002e44e6ba3817e66564c1581 (MD5) Previous issue date: 2016-03-15 / A Fibrose Pulmonar (FP), também denominada Alveolite Fibrosante, é uma pneumopatia idiopática, crônica e irreversível, que acomete o tecido pulmonar promovendo a deposição de tecido fibroso cicatricial e consequente remodelamento da arquitetura pulmonar. Considerando a inexistência de um tratamento clínico curativo que não seja apenas paliativo, a terapia com células-tronco desponta como alternativa promissora no tratamento da fibrose pulmonar e diversas outras patologias. Neste contexto, este projeto propôs o emprego da terapia celular com células-tronco mesenquimais de tecido adiposo humano (CTMTA) e “pool” de células mononucleares da medula óssea (BMMC), isoladas ou conjuntamente, em modelo experimental de fibrose pulmonar. Foram utilizados 36 ratos Wistar divididos em 6 grupos. O grupo controle recebeu instilação intratraqueal de tampão fosfato. Cinco grupos foram instilados com bleomicina (2U/animal) para a indução da doença e, após 14 dias, submetidos ou não a diferentes tratamentos utilizando células mononucleares da medula óssea e/ou células-tronco mesenquimais de tecido adiposo, sendo que um grupo recebeu tratamento placebo com tampão fosfato. A análise histológica evidenciou o desenvolvimento de fibrose nos animais instilados com bleomicina (p<0,0001) e uma melhora do quadro fibrótico nos animais submetidos ao tratamento, independentemente do tipo celular utilizado (p<0,001). A ventilação pulmonar basal revelou a presença de esforço respiratório nos animais instilados com bleomicina (p=0,0260), porém nenhum tipo de terapia foi capaz de reverter esse quadro. Não houve diferença significativa entre o perfil de leucócitos encontrados no lavado broncoalveolar dos animais sadios e doentes, entretanto a terapia com CTMTA promoveu a redução da porcentagem de linfócitos e o aumento de macrófagos nos animais doentes. O tratamento com BMMC e CTMTA, realizado de modo isolado ou em conjunto, mostrou remissão significativa do quadro de fibrose instalado. / Pulmonary Fibrosis (PF), also known as fibrosing alveolitis, is an idiopathic, chronic, irreversible lung disease that affects the lung tissue by promoting the deposition of fibrous scar tissue and subsequent lung architecture remodeling. Considering the need for curative medical treatment that is not only palliative, stem cell therapy is emerging as a promising alternative for the treatment of pulmonary fibrosis and many other diseases. ln this context, it is proposed in this study the cell therapy with mesenchymal stem cells derived from human adipose tissue (CTMTA) in association with the pool of mononuclear bone marrow cells (BMMC) in an experimental model of pulmonary fibrosis. The disease was induced in 4 groups of Wistar rats (n = 6) by intratracheal bleomycin (2U/animal). 14 days after instillation, each group was subjected to a specific treatment with BMMC and CTMTA, isolated or combined mode, while a fourth group was treated with phosphate buffer. The control group was instilled and treated with phosphate buffer. Histological analysis showed the development of disease in animais instilled with bleomycin (p<0,0001) and untreated, and improvement in the fibrous framework of animais subjected to cell therapy (p<0,001 ). The basal ventilation revealed the presence of respiratory effort in animais instilled with bleomycin, submitted or not to therapy (p=0,0260). Treatment with BMMC and CTMTA, performed alone or in conjunction mode showed similar results, since both promoted remission of pulmonary fibrosis.

Les premières utilisations du tissu adipeux comme produit de comblement en chirurgie plastique remontent à la fin du 19ème siècle. Depuis quelques décennies, la greffe de tissu adipeux a bénéficié d'un regain d'intérêt utilisant un procédé chirurgical rigoureux. Devant la démonstration de la survie cellulaire et les bons résultats cliniques obtenus, l'utilisation de cette technique s'est élargie à tous les domaines de la chirurgie plastique. Cette technique est simple et efficace et représente actuellement le meilleur moyen de restaurer les défauts de contours et de volume. Récemment, de nouvelles indications utilisant les capacitésrégénératrices du tissu adipeux ont été décrites. Elles concernent la cicatrisation des plaies chroniques et l'amélioration des dystrophies cutanées. Mais la limite de la greffe de tissu adipeux est l'absence de site donneur disponible au prélèvement. Le tissu adipeux est aujourd'hui reconnu comme la source la plus abondante de cellules souches mésenchymateuses multipotentes. Cela a donné un nouvel essor à la médecine régénérative pour réparer, remplacer ou régénérer les tissus et organes endommagés à partirdes cellules souches. Cette régénération se fait soit in-situ après administration des cellules souches, soit après développement in-vitro d'un tissu par ingénierie. Après une présentation du tissu adipeux et ses cellules souches ainsi que leurs applications actuelles en chirurgie plastique, le but de ce travail était de : 1. de clarifier les facteurs influençant les résultats de la greffe adipeuse pour une optimisation de cette technique. 2. d'explorer les possibilités offertes par les cellules souches adipeuses pour la médecine régénérative et l'ingénierie tissulaire, en vue d'une utilisation en chirurgie plastique / The first uses of adipose tissue as filler in plastic surgery started in the late 19th century. In recent decades, the adipose tissue transplantation has received renewed interest using a rigorous surgical procedure. Before the demonstration of cell survival and good clinical results, the use of this technique was extended to all areas of plastic surgery. This technique is simple and effective and is currently the best way to restore the defects of contour and volume. Recently, new indications using the regenerative capacity of adipose tissue have been described. They concern the healing of chronic wounds and the improvement of skin dystrophy. But the limit of the adipose tissue graft is the lack of available donor site for harvesting. Adipose tissue is now recognized as the most abundant source of multipotent mesenchymal stem cells. This gave a boost to regenerative medicine to repair, replace or regenerate damaged tissues and organs from stem cells. This regeneration is done either in-situ after administration of stem cells, after in-vitro development of tissue engineered. After a presentation of adipose tissue and stem cells and their current applications in plastic surgery, the aim of this study was to: 1. clarify the factors influencing the results of fat transplantation to optimize this technique. 2. explore the possibilities offered by ASCs for regenerative medicine and tissue engineering, for use in plastic surgery

L'accident vasculaire cérébral (AVC) est la principale cause de handicap acquis chez l'adulte. Au-delà de l'étroite fenêtre thérapeutique et des risques éventuels de la thrombolyse et de la thrombectomie mécanique, la thérapie cellulaire par cellules souches présente un fort potentiel. Plusieurs études ont montré que les cellules souches transplantées peuvent améliorer la récupération fonctionnelle après un AVC sur des modèles de rongeurs. L’imagerie multiparamétrique par résonance magnétique (IRM), qui inclue l'imagerie de diffusion et de perfusion, est aujourd’hui le protocole standard pour caractériser l'AVC. L'imagerie permet également de suivre in vivo les mécanismes sous-jacents de la thérapie cellulaire après un AVC de la phase aigüe à la phase chronique. Cependant, la quantification de l'hétérogénéité spatiale des lésions, clairement visible par IRM, reste un défi à l'heure actuelle. En effet, les techniques d'analyses d'images utilisées en routine sont basées sur le calcul des valeurs moyennes à partir de régions d'intérêts (ROI). Cette technique par ROI ne peut pas refléter l'hétérogénéité intra-lésionnelle. C'est pourquoi, de nouvelles stratégies d'analyses d'images doivent être développées et évaluées afin de quantifier l'hétérogénéité des lésions ischémiques mais aussi pour suivre l'évolution de cette hétérogénéité au cours du temps. Des approches utilisant des analyses par histogramme permettent d'évaluer l'hétérogénéité des lésions mais perdent l'information spatiale. Une alternative est l'utilisation d'une analyse d'image à l'échelle du voxel appelée "Parametric Response Map (PRM)". Cet outil a été décrit comme plus sensible que l'analyse par ROI dans le pronostic mais aussi dans le suivi thérapeutique chez des patients porteurs de tumeurs cérébrales ou encore atteints d'hémorragies cérébrales.Mon projet de thèse est divisé en deux parties: une étude préclinique chez le rat et une étude clinique (projet ISIS / HERMES). La première partie de ma thèse vise à évaluer les changements physiopathologiques mesurés par l'IRM après un traitement par cellules souches mésenchymateuses humaines (CSMh) sur un modèle d'AVC chez le rat. Des animaux présentant une occlusion transitoire de l'artère cérébrale moyenne (oACM) ou non (Sham) ont été traités ou non par une injection de CSMh. Au cours de cette étude, différents paramètres IRM ont été cartographiés en utilisant une IRM 7T (4 temps d'imagerie): le coefficient apparent de diffusion (ADC), le volume sanguin cérébral (CBV) et l'indice de taille des vaisseaux (VSI). Les cartes d'ADC, CBV et VSI ont été analysées en utilisant l'approche classique par ROI mais aussi par PRM. L'objectif de cette étude était de déterminer si l'analyse par PRM était capable de détecter plus précocement l'effet des CSMh que l'analyse par ROI. Durant la seconde partie de ma thèse, 6 paramètres IRM (imagerie de diffusion et de perfusion) ont été acquis chez 30 patients AVC. Les données IRM, analysées par valeur moyenne classique et par PRM, ont été corrélées avec des évaluations de la récupération fonctionnelle : le score NIHSS (National Institutes of Health Stroke Score) et l'échelle de Rankin modifiée (mRS) mesurés à différents temps post-ischémie. L’analyse PRM des cartes paramétriques IRM révèle des changements fins de la lésion et corrèle avec le pronostic à long terme après l’ischémie.En conclusion, la PRM pourrait être utilisée comme biomarqueur d’efficacité thérapeutique (combinaison d’images IRM et d’outils innovants d’analyse d'images) et comme biomarqueur pronostique des patients AVC. / Stroke is the leading cause of disability in adults. Beyond the narrow time window and possible risks of thrombolysis and mechanical thrombectomy, cell-therapies have strong potential. Reports showed that transplanted stem cells can enhance functional recovery after ischemic stroke in rodent models.To assess the mechanisms underlying the cell-therapy benefit after stroke, imaging is necessary. Multiparametric magnetic resonance imaging (MRI), including diffusion-weighted imaging (DWI) and perfusion-weighted imaging (PWI), has become the gold standard to evaluate stroke characteristics. MRI also plays an important role in the monitoring of cerebral tissue following stroke from the acute to the chronic phase. However, the spatial heterogeneity of each stroke lesion and its dynamic reorganization over time, which may be related to the effect of a therapy, remain a challenge for traditional image analysis techniques. To evaluate the effect of new therapeutic strategies, spatial and temporal lesion heterogeneities need to be more accurately characterized and quantified.The current image analysis techniques, based on mean values obtained from regions of interest (ROIs), hide the intralesional heterogeneity. Histogram-based techniques provide an evaluation of lesion heterogeneity but fail to yield spatial information. The parametric response map (PRM) is an alternative, voxel-based analysis technique, which has been established in oncology as a promising tool to better investigate parametric changes over time at the voxel level which concern the therapeutic response or prognosis of disease.The PhD project was divided into two parts: a preclinical and a clinical study. The goal of the first study was to evaluate the PRM analysis using MRI data collected after the intravenous injection of human mesenchymal stem cells (hMSCs) in an experimental stroke model. The apparent diffusion coefficient (ADC), cerebral blood volume (CBV) and vessel size index (VSI) were mapped using 7T MRI. Two analytic procedures, the standard whole-lesion approach and the PRM, were performed on data collected at 4 time points in transient middle cerebral artery occlusion (MCAo) models treated with either hMSC or vehicle and in sham animals. During the second PhD project, 6 MR parametric maps (diffusion and perfusion maps) were collected in 30 stroke patients (the ISIS / HERMES clinical trial). MRI data, analyzed with both a classic mean value and a PRM approaches, were correlated with the evaluation of functional recovery after stroke measured with the National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale (mRS) at 4 time points.In both studies, PRM analysis of MR parametric maps reveals fine changes of the lesion induced by a cell therapy (preclincal study) and correlate with long-term prognosis (clinical study).In conclusion, the PRM analysis could be used as an imaging biomarker of therapeutic efficacy and of prognostic biomarker of stroke patients.