La réaction du greffon contre l’hôte (GvHD) est une complication majeure de la transplantation de cellules souches hématopoïétiques (HSCT). Les traitements de prophylaxie contre le développement de la GvHD reposent essentiellement sur l’utilisation d’agents immunosuppresseurs, ce qui contribue à ralentir la reconstitution immunitaire post-greffe et à prolonger la durée de l’état immunosupprimé des patients. Le développement de prophylaxie pour la GvHD à base d’agents immunomodulateurs est ainsi privilégié. À l’aide d’un modèle murin xénogénique chez les souris NOD/scid-IL2rγ-/- (NSG), on a étudié le potentiel immunomodulateur des immunoglobulines intraveineuses (IVIG) dans la prévention de la GvHD, ainsi que leurs effets sur la qualité et la cinétique de la reconstitution immunitaire. On a déterminé qu’un traitement hebdomadaire d’IVIG peut effectivement réduire l’incidence de la GvHD, ainsi que la mortalité qui y est reliée, avec une efficacité similaire à celle obtenue avec la cyclosporine A, un immunosuppresseur couramment utilisé dans la prophylaxie de la GvHD. Par ailleurs, on a déterminé que le mécanisme d’action des IVIG dans la réduction de la GvHD est distinct de celui des immunosuppresseurs. De plus, on a démontré que les IVIG induisent l’expansion et l’activation des cellules NK présentes au sein du greffon, lesquelles sont nécessaires pour l’obtention de l’effet protecteur des IVIG contre le développement de la GvHD, et sont dépendantes de la présence de lymphocytes T activés. Grâce à un modèle murin humanisé, on a également démontré que le traitement hebdomadaire d’IVIG induit un délai transitoire de la reconstitution humorale, ce qui n’affecte toutefois pas la qualité globale de la reconstitution immunitaire. Ces résultats mettent cependant en doute la pertinence de l’utilisation des IVIG dans les protocoles cliniques de prophylaxie de la GvHD, puisque les immunosuppresseurs seront toujours utilisés, et qu’on a démontré que les IVIG ont besoin de lymphocytes T activés afin de prévenir efficacement le développement de la GvHD. / Graft-versus-Host Disease (GvHD) is a major complication following hematopoietic stem cell transplantation (HSCT). Prophylactic treatments for the prevention of GvHD rely mostly on the use of immunosuppressors, which contribute to inhibit the patient’s immune reconstitution and prolong their immunosuppressed state. Development of immunomodulator-based prophylactic treatments is therefore preferred. Using NOD/scid-IL2rγ-/- mice, we developed a xenogeneic mouse model to assess the immunomodulatory potential of intravenous immunoglobulins (IVIG) for the prevention of GvHD, along with assessing their effect on the kinetics and the quality of the immune reconstitution in mice. We determined that weekly IVIG treatments reduced the incidence of GvHD and its related mortality. The effectiveness of IVIG for the prevention of GvHD was similar to that of cyclosporine A, an immunosuppressive drug routinely used for the prophylactic treatment of GvHD. Furthermore, we demonstrated that IVIG has a mechanism of action that is different from that of immunosuppressors. IVIG induce the expansion and activation of NK cells from the graft, which is mandatory for the preventive effect of IVIG on GvHD development. Furthermore, this IVIG-induced expansion and activation of NK cells require the presence of activated T lymphocytes. Using our humanized mouse model, we have also demonstrated that weekly IVIG treatments cause a transient delay of the humoral reconstitution, but do not affect the overall quality of the immune reconstitution. We have demonstrated that activated T lymphocytes are mandatory for the effective expansion and activation of NK cells, which in turn are essential to the IVIG-induced prevention of GvHD, and immunosuppressors will always be part of the prophylactic regimen of GvHD, therefore shining a doubt on the usefulness of adding IVIG to the prophylactic treatments for the prevention of GvHD.

Streptococcus thermophilus est la bactérie lactique la plus utilisée après Lactococcus lactis dans l'industrie laitière pour la fabrication de yaourts et de fromages à pâte cuite (Emmental, gruyère), filée (Mozarella) ou pressée (Cheddar). Il s'agit du seul streptocoque à avoir le statut de bactérie GRAS (Generally Recognized As Safe). Dans un premier temps le potentiel probiotique de 30 souches de S. thermophilus de différentes origines a été testé par l'étude de leurs capacités à résister aux différentes conditions de stress rencontrées pendant leur passage dans le tractus gastro-intestinal (bas pH, sels biliaires et stress oxydant), de leur capacité à adhérer aux cellules épithéliales intestinales et de leurs propriétés immunomodulatrices. La majorité des souches réduit la production d'interleukine IL-8 (pro-inflammatoire) alors qu'elles induisent la production d'interleukine IL-10 (anti-inflammatoire) et l'IL-12 (pro-inflammatoire). Sur la base du rapport IL-10/IL-12, qui permet d'apprécier le potentiel anti-inflammatoire d'une souche, nous avons observé que certaines d'entre-elles pourraient avoir un fort potentiel anti-inflammatoire. L'Analyse en Composantes Principales (ACP) nous a permis de séparer les souches en 6 catégories différentes présentant des propriétés distinctes. A l'intérieur de chaque classe, une variabilité entre les souches a été observée et des caractéristiques intéressantes identifiées. Cependant, aucune des classes ne peut être considérée comme contenant le probiotique « parfait ». Suite à cette étude et sur la base de sa résistance aux stress gastriques et de ses capacités d'adhésion, et sachant que la séquence de son génome est disponible, la souche LMD-9 a été sélectionnée pour la deuxième partie de ce travail, à savoir la construction d'un outil basé sur la technologie R-IVET pour étudier l'état physiologique de S. thermophilus dans le tractus digestif. L'outil R-IVET mis au point se compose de deux éléments: un vecteur plasmidique portant le gène cre codant une recombinase spécifique dépourvu de son promoteur et une cassette chromosomique composée d'un gène de résistance à la spectinomycine flanqué par des sites loxP, reconnus par la recombinase Cre. La fonctionnalité de l'outil R-IVET a ensuite été testée par le clonage de trois promoteurs différents de S. thermophilus (PprtS, Pshsp and Plac) en amont de cre. Le système a été valide in vitro avec les trois promoteurs et in vivo avec le promoteur Plac / Streptococcus thermophilus is a lactic acid bacterium used after Lactococcus lactis in the dairy industry for the production of yogurt and cheeses like Emmental, Gruyere, Mozarella and Cheddar. It is the only streptococcus to have the GRAS (Generally Recognized As Safe) status. In this work, the probiotic potential of 30 S. thermophilus strains from different origins was tested by studying their ability to resist different stress conditions encountered during their passage through the GIT (low pH, bile salts and oxidative stress), their ability to adhere to intestinal epithelial cells and their immunomodulatory properties. Majority of the strains reduced the production of interleukin IL-8 (pro-inflammatory) and induced the production of interleukin IL-10 (anti-inflammatory) and IL-12 (pro-inflammatory). On the basis of the ratio IL-10/IL-12 which allows to evaluate the anti-inflammatory potential of a probiotic, several strains appeared to have a high the anti-inflammatory potential. Principal Component Analysis (PCA) allowed us to classify strains in 6 different categories with different properties. Within each class, variability and interesting features were observed, but none of the classes could be considered as containing the perfect probiotic. Following this study and on the basis of its resistance to gastric stress and its adhesion capacity and knowing that its genome sequence is available, the strain LMD-9 was selected for the second part of this work, namely the construction of a tool based on R-IVET to study the physiological state of S. thermophilus in the digestive tract. This tool is composed of two elements: a plasmid vector (pULNcreB), carrying the gene cre encoding a site-specific recombinase without its promoter and a chromosomal cassette composed of a gene of resistance to spectinomycin flanked by loxP sites which are recognized by the recombinase Cre. The functionality of the tool R-IVET was then tested by cloning three different promoters of S. thermophilus (PprtS, Pshsp and Plac) upstream of cre. System was valid in vitro with all the three promoters and in vivo by using the lactose operon promoter Plac

In this study, we examined the immunomodulatory effect of excretory/secretory products, crude adult extracts and crude larvae extracts from Hymenolepis diminuta on the intestinal epithelilal cell line from a rat. For determination of the immunomodulation effect of all H. diminuta extracts was used relative gene expression of TNFa, IL-17re and IL-33 from epithelial cells and it was tested using real-time PCR. Our result showed that excretory/secretory products had the strongest antiinflammatory effect on the epithelial cells. We assume that crude adult extracts play an important role in increase of gene expression of IL-33 and also in the immunomodulatory ability of H. diminuta in the host organism.

Made available in DSpace on 2015-07-22T20:50:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-22. Added 1 bitstream(s) on 2015-08-11T03:26:13Z : No. of bitstreams: 1 Publico-12887.pdf: 1856396 bytes, checksum: 42de9f60d780e16634f25ebf960a3c24 (MD5) / Lesões no sistema nervoso central (SNC) levam a permeabilidade da barreira hematoencefálica, o que permite a entrada de células do sistema imune e a ativação das células da glia, principalmente microglia e astrócitos. Esse processo desencadeia a secreção de mediadores inflamatórios por essas células. As citocinas são as principais moléculas da resposta neuroinflamatória e são críticas para a regulação desta resposta, exercendo uma variedade de ações no SNC. Células tronco mesenquimais (CTMs), que possuem potencial proliferativo e são capazes de originar linhagens celulares distintas e especializadas, também secretam essas moléculas, caracterizando um poder imunomodulador. As CTMs, particularmente as derivadas da medula óssea, promovem o reparo tecidual pela secreção de fatores que aumentam a regeneração do tecido, estimulando proliferação, migração e diferenciação de progenitores endógenos encontrados na maioria dos tecidos, diminuindo a resposta imune e inflamatória e a apoptose. A habilidade de essas células alterarem o microambiente através de sua influência trófica pode contribuir mais significativamente para o reparo do tecido que a transdiferenciação. Nossa hipótese é que as citocinas secretadas pelas CTMs poderiam participar da atração de células tronco neurais endógenas para um local de lesão no SNC, criando um microambiente favorável para essas células. Tendo isso em vista, esta tese teve como objetivo estudar os efeitos dos fatores secretados pelas CTMs sobre células tronco neurais (CTNs) in vitro, e analisar a expressão de citocinas por CTMs in vivo em um modelo de lesão traumática no SNC. Primeiramente, avaliamos os efeitos dos fatores secretados pelas CTMs sobre apoptose, proliferação e diferenciação de CTNs adultas derivadas da zona subventricular e cultivadas como neuroesferas. Para isso, cultivamos as neuroesferas em meio condicionado por CTMs derivadas de medula óssea. Além disso, foram realizadas lesões no córtex motor primário dos animais, seguidas da injeção de CTMs no local da lesão. Nossos resultados indicam que os fatores secretados pelas CTMs não induzem nem previnem a apoptose das CTNs, aumentam a proliferação dessas células e induzem maior expressão do gene GFAP in vitro, o que indicaria uma tendência a diferenciação em astrócitos. Nos experimentos in vivo, nossos resultados mostram que a injeção das CTMs em um modelo de lesão aguda no SNC diminui a expressão de citocinas pró-inflamatórias no tecido lesado, indicando que os fatores solúveis secretados por CTMs podem modular a inflamação no local lesado, o que pode ser interessante para a criação de um microambiente favorável para CTNs endógenas e conseqüentemente para o reparo do tecido lesado. / Central nervous system (CNS) injury breakes the impermeability of the blood brain barrier, this allows the invasion of immune cells and activation of glial cells, mainly microglia and astrocytes. This process triggers the secretion of inflammatory mediators by these cells. Cytokines are the main molecules in neuroinflammatory response and are critical for its regulation, exerting a variety of actions in the CNS. Furthermore, mesenchymal stem cells (MSC) which have proliferative potential and are able to originate different and specialized cell lineages, also secrete these molecules, characterizing its immunomodulatory function. MSC, particularly those derived from bone marrow, promote tissue repair by secreting factors that enhance tissue regeneration stimulating proliferation, migration and differentiation of endogenous stem-like progenitors found in most tissues, decreasing inflammatory and immune reactions and apoptosis. The ability of such cells to alter tissue microenvironment through its trophic influence may contribute more significantly than their capacity for transdifferentiation in effecting tissue repair.Our hypothesis is that MSC secreted cytokines could take part in the attraction of endogenous neural stem cells (NSC) to an injury site in the CNS, providing a favorable microenvironment for these cells. Our aim was to study the effects of factors secreted by MSC on NSC in vitro and to analyse the MSC cytokines expression in vivo in a model of CNS traumatic injury. We first evaluated the effects of MSC secreted factors on apoptosis, proliferation and differentiation of adult NSC derived from the subventricular zone and cultured as neurospheres. Neurospheres were cultured in MSC conditioned medium (MSC-CM), which was obtained from bone marrow-derived MSC cultures. Besides a traumatic injury was performed at the primary motor cortex of mice and MSCs were injected at the injury site. Our results show that MSC secreted factors do not induce or prevent NSC apoptosis, increase NSC proliferation and induce bigger expression of GFAP gene in vitro, this could indicate a tendency of differentiation to astrocytes. In vivo experiments show that MSC injection at an acute model of injury diminishes pro-inflamatory cytokines in the injured tissue, suggesting that MSC secreted factors may modulate the inflammation at the injury site, which may be interest to the development favorable microenvironment for endogenous NSC and consequently repair of the injured tissue. / TEDE / BV UNIFESP: Teses e dissertações

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Orf virus (ORFV) belongs to the family Poxviridae, subfamily Chordopoxvirinae and genus Parapoxvirus and is the agent of contagious ecthyma, a mucocutaneous disease that affects mainly young sheep and goats and, occasionally, may affect people. The clinical lesions progress through stages of hyperemia, papules, vesicles, pustules, ulcers and proliferative and scabby lesions, located mainly on the labial commissure, lips and nostrils. Variable clinical lesions with different degrees of severity often occur in sheep and goats, and may be associated with host and/or viral genetics. The present study aimed to investigate the phenotype in vivo and to characterize virulence genes of four ORFV isolates recovered from contagious ecthyma outbreaks in Rio Grande do Sul State, Brazil. Twenty sheep, aged 6 and 8 months, were divided into five groups of four animals each and inoculated in the labial commissure with homogenates of scabs (viral titers 105.6TCID50/ml) obtained from different outbreaks: SV269/11, SV252/11, SV581/11 and SV820/10-Canguçu. The animals were evaluated for 30 days in clinical and virological aspects by clinical inspection and swab collection for virus isolation. A clinical score was established for each animal and group. All ORFV inoculated animals developed classical ecthyma contagious lesions, characterized by hyperemia, papules, macules, vesicles, pustules and scabs in varied degrees and duration. SV269/10 and SV820/10-Canguçu isolates induced more severe lesions resulting in higher clinical scores and longer duration of lesions. The animals inoculated with SV581/11 developed milder lesions and clinical scores significantly lower than other groups, but they shed virus for a longer period of time. For genetic analyses, PCR amplification and nucleotide sequencing of three virulence genes (VEGF, VIR and IL-10v) were performed. Deletion and mutations on VEGF and IL-10v amino acid sequence of SV581/11 and SV252/11 isolates were identified. The degree of amino acid identity among ORFV sequences was variable, and the lowest homology was found in the VEGF gene of SV581/11 when compared with the standard strains and other viruses. Thus, the present results showed that SV581/11 and SV252/11 isolates, particularly the former, are less virulent in sheep than SV269/11 and SV820/10-Canguçu. Possibly, the variable phenotypic observed in vivo is due to genetic alterations detected in the analyzed virulence genes. / O vírus da orf (ORFV) pertence à família Poxviridae, subfamília Chordopoxvirinae gênero Parapoxvirus e é o agente etiológico do ectima contagioso, uma doença mucocutânea que afeta principalmente ovinos e caprinos jovens e pode, ocasionalmente, afetar pessoas. Clinicamente, a enfermidade evolui com a formação de áreas hiperêmicas, vesículas, pústulas, úlceras e lesões proliferativas e crostosas sobre a pele dos lábios, comissura labial e narinas. Apresentações clínicas variáveis, com diferentes graus de severidade, ocorrem frequentemente e podem estar associadas a características do hospedeiro e, principalmente, a características genéticas do agente. O presente trabalho teve como objetivo investigar o fenótipo in vivo e caracterizar genes de virulência de quatro amostras de ORFV oriundas de surtos no Estado do Rio Grande do Sul, Brasil. Para isso, foram utilizados vinte ovinos, com idade entre 6 e 8 meses, divididos em cinco grupos de quatro animais cada. Os ovinos foram inoculados na comissura labial com homogeneizados de crostas (títulos virais 105,6 DICC50/ml) obtidas dos surtos (amostras SV269/11, SV252/11, SV581/11 e SV820/10-Canguçu). Os animais foram avaliados durante 30 dias com relação aos aspectos clínicos e virológicos, por inspeção clínica e coleta de suabes das lesões para detecção da excreção viral. As manifestações clínicas foram convertidas em um escore clínico, para cada animal e para os grupos. Os animais inoculados com os as quatro amostras desenvolveram lesões típicas de ectima contagioso, caracterizadas por hiperemia, pápulas, máculas, vesículas e pústulas, e formação de crostas em diferentes graus de intensidade e duração. As amostras SV269/10 e SV820/10-Canguçu induziram lesões mais graves, escores clínicos maiores e maior tempo de duração das lesões. Os animais inoculados com a amostra SV581/11 desenvolveram lesões e escores clínicos significativamente inferiores aos demais grupos, mas excretaram o vírus por período mais longo. Para a caracterização genética, foi realizada a amplificação por PCR e sequenciamento de nucleotídeos de três genes de virulência (VEGF, VIR e IL-10v). Foram identificadas deleções e mutações nas sequências dos genes VEGF e IL-10v das amostras SV581/11 e SV252/11. O grau de identidade de aminoácidos entre as amostras foi variável, sendo que a menor homologia foi encontrada no gene VEGF da amostra SV581/11, quando comparado com os demais vírus e a cepa padrão. Assim, os resultados obtidos demonstram que as amostras SV581/11 e SV252/11, em especial a primeira, foram menos virulentas em ovinos do que as amostras SV269/11 e SV820/10-Canguçu. Possivelmente essa diferença fenotípica observada in vivo seja resultado das alterações genéticas detectadas nos genes de virulência.

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PhD (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal Universtiy of Technology / The genus Tulbaghia has been used in traditional medicine to treat various ailments such as fever, earache, tuberculosis and esophageal cancer. However, there is limited scientific evidence to support its use. Therefore the objectives of this study were to perform phytochemical analysis, investigate the antioxidant, antimicrobial, anticancer, immunomodulatory activities and toxicity of crude acetone and water extracts from selected Tulbaghia species. Standard methods were used for preliminary phytochemical analysis. The total phenolic content of the plant extracts was determined using the folin ciocalteu method whereas the total flavonoids were determined by using the aluminium chloride colorimetric method. DPPH and ABTS assays were used to evaluate the antioxidant activity. The antimicrobial activity was assessed by agar well diffusion, microtiter dilution and time kill assays. For anticancer studies, the antiproliferative activity of the extracts was evaluated using the MTT assay on Hkesc-1 and KB cells. Morphological changes of the cancer cells treated with extracts were examined using light microscopy. Induction of apoptosis was assessed using fluorescence microscopy and acridine orange/ethidium bromide staining. Flow cytometry analysis was conducted to examine the multicaspase activity and cell cycle arrest. For immunomodulatory activity, the Greiss reagent and Luminex cytokine assays were used to determine the effect of the extracts on NO production and the concentration of the cytokines in the treated cells, respectively. Toxicity of selected Tulbaghia species was examined by investigating the effect of the extracts on the metabolic activity and cell membrane integrity on the treated RAW264.7 cells using the MTT and LDH assays, respectively. The zebrafish assay was used to evaluate the embryotoxicity and teratogenic effects of crude acetone and water extracts of T. violacea at 24 h intervals for 96 h post fertilisation (hpf). The percentage mortality, hatchability and heart rate were examined. Phytochemical screening of eight Tulbaghia species demonstrated the presence of flavonoids, glycosides, tannins, terpenoids, saponins and steroids. The amount of total phenol and flavonoid content varied in different plant extracts ranging from 4.50 to 11.10 milligrams gallic acid equivalent per gram (mg GAE/g) of fresh material and 3.04 to 9.65 milligrams quercetin equivalent per gram (mg QE/g) of fresh material respectively. The IC50 values based on DPPH and ABTS for T. alliacea (0.06 and 0.06 mg/mL) and T. violacea (0.08 and 0.03 mg/mL) were generally lower showing potential antioxidant activities. For antimicrobial activity, the acetone extracts of T. acutiloba, T. alliacea, T. leucantha, T. ludwigiana, T. natalensis and T. simmleri showed moderate antimicrobial activity against all test organisms while the water extracts showed moderate to no activity. One species, T. cernua, showed poor activity against all the tested microbes. The acetone and water extracts of T. violacea showed the greatest antibacterial and antifungal activity against all the tested microorganisms with minimum inhibitory concentration ranging from 0.1 mg/mL to 3.13 mg/mL. The acetone extracts of T. violacea also exhibited both bacteriostatic/fungistatic and bactericidal/fungicidal activity depending on the incubation time and concentration of the extract. The bactericidal/fungicidal activity was observed at x2 MIC. The results for anticancer activity showed that treatment of Hkesc-1 cells with acetone and water crude extracts had anti-proliferative activity with IC50 values of 0.4 mg/mL and 1.625 mg/mL, respectively while KB had 0.2 mg/mL and 1 mg/mL, respectively. Morphological changes such as blebbing, cell shrinkage and rounding were observed in the treated cells suggesting that apoptosis was taking place. AOEB staining showed that the level of apoptosis was dependent on the concentration of the extracts. The activation of multicaspase activity in both Hkesc-1 and KB treated cells was also concentration dependent leading to cell death by apoptosis and the induction of cell cycle arrest at the G2/M phase. Immunomodulatory activity results indicated that cell viability was above 80% when concentrations of 50 µg/mL or less of both acetone and water crude was used. Treatment with the acetone extract had no significant effect (p>0.05) on the LPS induced NO production in RAW264.7 cells except at 50 µg/mL where significant inhibition was observed. The water extract had no significant effect (p>0.05) on NO production at all the concentrations. Treatment of LPS–induced RAW264.7 cells with acetone extract stimulated the production of IL-1α, IL-6 and TNF-α, but had no significant effect (p > 0.05) on IL-1β. On the other hand, treatment with the water extracts stimulated the production of IL-1α, IL-6 but had no significant effect (p>0.05) on TNF-α and IL-1β. Treatment of LPS-induced RAW264.7 cells with the acetone extract had very little stimulatory effect on IL-4, IL-5 and IL-13 and no significant effect on IL-10 whereas for the water extract a significant stimulatory effect was only observed for IL-4 after 48 h of treatment. High concentrations (>10000 pg/mL) of MCP-1, MIP1-α, MIP1-β, MIP-2, GCSF, GM-CSF, RANTES and IP-10 were also observed in acetone and water extract treated RAW264.7 cells. For toxicity studies, acetone and aqueous crude leaf extracts from T. alliacea, T. simmleri, and T. violacea had a significant inhibitory (p<0.05) effect on the RAW264.7 cells after 48h treatment. Acetone extracts from T. alliacea, T. simmleri and T. violacea resulted in IC50 values of 0.48 mg/mL, 0.72 mg/mL and 0.1 mg/mL, respectively. Treatment with water extracts showed minimal toxic effect indicated by higher IC50 values of 0.95 mg/mL, 2.49 mg/mL and 0.3 mg/mL for T. alliacea, T. simmleri and T. violacea, respectively. The LDH release by macrophages after 24 h treatment with acetone extracts was observed to be concentration dependent while treatment with water extracts did not induce LDH release. The zebra fish assay showed a lethal dose (LD50) for the T. violacea acetone crude extract of 20 μg/mL whereas that for water extract was 85 μg/mL. The observed teratogenic effects included scoliosis, edema of the pericardial cavity, retarded yolk resorption, hook-like/bent tail and shorter body length. In conclusion, the results from this study indicate that the extracts from the eight Tulbaghia species examined contain phytochemicals that may have the antioxidant, antimicrobial, anticancer and immunomodulatory properties. Extracts from T. violacea were observed to be the most potent. This study thus supports the use of T. violacea in treating bacterial and fungal infections in traditional medicine. The results of this study also confirm the anticancer potential of T. violacea. The immunomodulatory activity of the acetone and water extracts from T. violacea indicated a dominantly pro-inflammatory activity. Traditional medicine prepared form T. violacea may be of benefit to individuals with weak immune systems. The toxicity of selected Tulbaghia species was observed to be concentration, extract and time dependent. Therefore, traditional medicine prepared from Tulbaghia extracts should be taken with caution preferably in small doses over a short period of time. Future studies will focus on the identification of the bioactive compound(s) responsible for the antimicrobial, anticancer and immunomodulatory activities.

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[ES] Las células mesenquimales estromales (MSC) poseen una serie de cualidades inmunológicas, pro-angiogénicas y regenerativas que las convierten en un excelente candidato para el tratamiento de diversas patologías. A pesar de las pruebas contundentes obtenidas en modelos preclínicos que demuestran la actividad terapéutica de las MSC, los ensayos clínicos no han podido mostrar hasta ahora un beneficio consistente, probablemente debido a deficiencias metodológicas y a la falta de estandarización, así como a la variabilidad genética intrínseca a los estudios en humanos. Debido a ello, ha sido necesario profundizar en los mecanismos responsables del beneficio terapéutico y rediseñar las estrategias clínicas. En los últimos años, se ha observado que la reparación tisular mediada por las MSC se produce de forma paracrina, y se ha constatado que las vesículas extracelulares (EVs) secretadas por las MSC (EVMSC) son capaces de recapitular las propiedades inmunosupresoras de las células parentales. Además, las estrategias terapéuticas basadas en vesículas tienen grandes ventajas en términos de bioseguridad y producción en condiciones de grado clínico, reduciendo significativamente el coste de dichas terapias. Sin embargo, la dosis efectiva en grandes mamíferos, incluidos los humanos, es bastante elevada y la producción industrial de EVs se ve dificultada en parte, por la senescencia proliferativa que afecta a las MSC durante la expansión celular masiva. En este trabajo hemos intentado solventar los principales escollos de la terapia con EVs incrementando su potencial inmunosupresor y reduciendo por tanto la dosis efectiva. Este incremento se ha conseguido gracias a la sobreexpresión del factor inducible por hipoxia 1-alpha y al desarrollo de un medio de acondicionamiento en cultivo basado en citoquinas. Además, la inmortalización de las células secretoras mediante la transducción del gen de la telomerasa humana ha permitido tanto la estandarización del producto como su producción a gran escala. La eficacia de estas EVs ha sido testada en diferentes poblaciones celulares in vitro: linfocitos T, monocitos, células Natural Killer, macrófagos, células endoteliales y fibroblastos; y en dos modelos de ratón: hipersensibilidad retardada y colitis aguda inducida por TNBS. En conclusión, hemos desarrollado una fuente de EVs de larga duración que secreta grandes cantidades de vesículas con mayor capacidad inmunosupresora y antiinflamatoria, facilitando un producto terapéutico más estándar y fácil de producir para el tratamiento de enfermedades inflamatorias inmunomediadas. / [CA] Les cèl·lules mesenquimals estromals (MSC) posseeixen una sèrie de qualitats immunològiques, pro-angiogèniques i regeneratives que les converteixen en un excel·lent candidat per al tractament de diverses patologies. Tot i les proves contundents obtingudes en models preclínics que demostren l'activitat terapèutica de les MSC, els assaigs clínics no han pogut mostrar fins ara un benefici consistent, probablement degut a deficiències metodològiques i a la manca d'estandardització, així com a la variabilitat genètica intrínseca als estudis en humans. A causa d'això, ha calgut aprofundir en els mecanismes responsables del benefici terapèutic i redissenyar les estratègies clíniques. En els últims anys, s'ha observat que la reparació tissular intervinguda per les MSC es produeix de forma paracrina, i s'ha constatat que les vesícules extracel·lulars (EVs) secretades per les MSC (EVMSC) són capaços de recapitular les propietats immunosupressores de les cèl·lules parentals. A més, les estratègies terapèutiques basades en vesícules tenen grans avantatges en termes de bioseguretat i producció en condicions de grau clínic, reduint significativament el cost d'aquestes teràpies. No obstant això, la dosi efectiva en grans mamífers, inclosos els humans, és bastant elevada i la producció industrial de les EVs es veu dificultada en part, per la senescència proliferativa que afecta les MSC durant l'expansió cel·lular massiva. En aquest treball hem intentat solucionar els principals esculls de la teràpia amb EVs incrementant el seu potencial immunosupressor i reduint per tant la dosi efectiva. Aquest increment s'ha aconseguit gràcies a la sobreexpressió del factor induïble per hipòxia 1-alpha i a el desenvolupament d'un mitjà de condicionament en cultiu basat en citoquines. A més, la immortalització de les cèl·lules secretores mitjançant la transducció del gen de la telomerasa humana ha permès tant l'estandardització del producte com la seva producció a gran escala. L'eficàcia d'aquestes EVs ha estat testada en diferents poblacions cel·lulars in vitro: limfòcits T, monòcits, cèl·lules Natural Killer, macròfags, cèl·lules endotelials i fibroblasts; i en dos models de ratolí: hipersensibilitat retardada i colitis aguda induïda per TNBS. En conclusió, hem desenvolupat una font de EVs de llarga durada que secreta grans quantitats de vesícules amb major capacitat immunosupressora i antiinflamatòria, facilitant un producte terapèutic més estàndard i fàcil de produir per al tractament de malalties inflamatòries inmunomediades. / [EN] Mesenchymal stromal cells (MSC) possess several immunological, pro-angiogenic and regenerative qualities that make them an excellent candidate for the treatment of various pathologies. Despite compelling evidence from preclinical models demonstrating the therapeutic activity of MSCs, clinical trials have so far failed to show consistent benefit, probably due to methodological shortcomings and lack of standardisation, as well as the genetic variability intrinsic to human studies. As a result, it has been necessary to further investigate the mechanisms responsible for therapeutic benefit and to redesign clinical strategies. In recent years, it has been observed that MSC-mediated tissue repair occurs in a paracrine pathway, and it has been confirmed that extracellular vesicles (EVs) secreted by MSC (EVMSC) are able to recapitulate the immunosuppressive properties of the parental cells. Moreover, vesicle-based therapeutic strategies have great advantages in terms of biosafety and production under clinical-grade conditions, significantly reducing the cost of such therapies. However, the effective dose in large mammals, including humans, is quite high and the industrial production of EVs is hampered in part by the proliferative senescence that affects MSC during massive cell expansion. In this work, we have attempted to overcome the main challenges of EVs therapy by increasing their immunosuppressive potential and thus reducing the effective dose. This increase has been achieved by overexpression of hypoxia-inducible factor 1-alpha and the development of a cytokine-based culture conditioning medium. In addition, immortalization of secretory cells by transduction with the human telomerase gene has allowed both product standardisation and large-scale production. The efficacy of these EVs has been tested in different cell populations in vitro: T lymphocytes, monocytes, Natural Killer cells, macrophages, endothelial cells and fibroblasts; and in two mouse models: delayed-type hypersensitivity and TNBS-induced acute colitis. In conclusion, we have developed a long-lasting source of EVs that secretes large amounts of vesicles with enhanced immunosuppressive and anti-inflammatory capacity, providing a more standard and easier-to-produce therapeutic product for the treatment of immune-mediated inflammatory diseases. / Gómez Ferrer, M. (2022). Terapias innovadoras basadas en vesículas extracelulares derivadas de células madre mesenquimales modificadas genéticamente [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182560