Microglia have the potential to produce specific neurotrophic molecules in response to injury and brain diseases. Activated microglia are seen after brain injury or in neurological disease, such as Parkinson¡¦s disease (PD). PD is a progressive neurodegenerative disorder. Although its cause remains unknown, it is believed that enhanced oxidative stress is a major component in the pathogenesis of nigral cell death in PD. Previous results have shown that DA induces apoptosis of dopaminergic neurons in a time- and concentration- dependent manner. In addition, a number of studies have shown that Zn2+ may enter the cell to reach toxic concentrations and that Zn2+ concentration is higher in the striatum of the postmortem brains of PD patients than that of the control brains. We have previously shown that Zn2+ synergistically enhanced the dopamine- and H2O2- induced PC12 cell death. To study the role of microglia in the cell death, I have examined the effect of conditioned medium from a human microglia cell line on the PC12 cell death induced by dopamine and Zn2+. The result shows that conditioned medium inhibits the PC12 cell death and the phosphorylation of JNK induced by dopamine and Zn2+ is diminished by the conditioned medium. It appears that the factor(s) that are responsible for the protection is heat-stable because the conditioned medium heated in 70¢Jfor 30 minutes still has the ability to protect the cell death. Cell death induced by A23187 and C2-ceramide, but not by staurosporine can be protected by the conditioned medium. Results from this study suggest that the microglia secrete some factors which can protect neuron.

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<p>The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium.</p><p>Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex.</p>

Exogenous and endogenous cannabinoids have been reported to modulate functional activities of macrophages. It is recognized that macrophages express primarily the CB2 cannabinoid receptor, but recent studies indicate that its expression is differential in relation to activation state with maximal levels occurring when cells are in "responsive" and "primed" states. The functional activities of macrophages when in these states of activation are the most susceptible to the action of cannabinoids, at least in terms of a functional linkage to the CB2. To assess the effect of cannabinoid treatment on macrophage chemotaxis and test the hypothesis that cannabinoids inhibit the chemotactic response of macrophages and microglia to endogenous and exogenous, pathogen-derived stimuli, primary murine peritoneal macrophages and neonatal rat microglia were used. Chemotaxis assays and scanning electron microscopy studies demonstrated that cannabinoids inhibit chemotaxis, a signature activity attributed to "responsive" macrophage-like cells, to the endogenous chemokine RANTES (Regulated upon Activation Normal T-cell Expressed and Secreted) and to Acanthamoeba conditioned medium containing secreted proteases. The partial agonist delta-9-tetrahydrocannabinol (THC), administered in vitro, inhibited the chemotactic response of peritoneal macrophages to the chemokine RANTES and to Acanthamoeba conditioned medium. In vivo treatment with THC also resulted in inhibition of the in vitro chemotactic response of murine peritoneal macrophages to RANTES and amoebic conditioned medium. Pharmacological studies employing cannabinoid receptor agonists and antagonists demonstrated the involvement of CB2 in cannabinoid-mediated inhibition of peritoneal macrophage chemotaxis to RANTES and Acanthamoeba conditioned medium, implying that signaling through cannabinoid receptors may desensitize chemokine receptors. Treatment with cannabinoids had no apparent effect on chemokine receptor mRNA levels, but did enhance CCR5 protein phosphorylation. Macrophage migration to Acanthamoeba conditioned medium may involve activation and signaling through protease activated receptors (PARs), as pathogen-derived proteases have been shown to activate PARs and initiate cellular migration; however, further studies are required to demonstrate PAR activation by amoebic conditioned medium and to assess the effects of cannabinoids on PAR signaling. Acanthamoeba are opportunistic pathogens that cause Granulomatis amoebic encephalitis, an infection of the CNS that is often fatal. THC treatment has been shown to increase mortality to Acanthamoeba infections and is characterized by an absence of granuloma formation. We hypothesize that inhibitory effect of THC on macrophage migration may be a key factor in cannabinoid-mediated immunosuppression. To assess the effect of cannabinoids on microglial migration to Acanthamoeba conditioned medium, chemotaxis assays were performed using primary rat microglia treated with cannabinoids. These studies demonstrated that cannabinoids inhibit microglial chemotaxis to amoebic conditioned medium. Furthermore, the studies demonstrate that cannabinoids, acting through cannabinoid receptors, may cross-talk with a diverse array G-protein coupled receptors so as to modulate responsiveness of macrophage and macrophage-like cells.

Orientador: Fernanda da Cruz Landim / Resumo: As células-tronco mesenquimais (MSCs) exercem seus efeitos terapêuticos predominantemente pela sua atividade parácrina. Como o secretoma desempenha um papel direto nas atividades biológicas das MSCs, a análise de seus componentes proteicos é um passo fundamental para identificar os principais responsáveis no controle e regulação dos muitos processos biológicos desenvolvidos por essas células. O secretoma de MSCs pode ser modificado e melhorado de forma in vitro para estimular efeitos celulares específicos desejados para aplicações terapêuticas, e uma maneira de modificá-lo é por meio de modificações nas condições de cultura. No presente estudo, objetivou-se descrever o secretoma de células-tronco mesenquimais derivadas de tecido adiposo (AD-MSCs) de gatos e cães submetidos a diferentes condições de cultivo, identificamos e comparamos os efeitos exercidos por diferentes modificações de cultivo. Para a produção de meio condicionado, as células foram descongeladas e cultivadas com meio DMEM/F12 com 20% de FBS, suplementado com antibióticos e antimicóticos em uma incubadora com umidade controlada a 37,5 °C, e 5% de CO2. Após atingir pelo menos 70% de confluência, as garrafas foram lavadas três vezes com solução salina balanceada Hanks (HBSS) e foram cultivadas em quatro condicionamentos distintos. O meio condicionado foi colhido após 4 dias, centrifugado por 10 min/300 g, filtrado em um filtro 0,22 μm e congelado a -80 °C. Foram realizadas a extração e concentração proteica de to... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: It is now known that mesenchymal stem cells (MSCs) exert their therapeutic effects predominantly through their paracrine activity. Since the secretome plays a direct role in the biological activities of MSCs, the analysis of their protein components is a fundamental step in identifying the main responsible for the control and regulation of the many biological processes developed by these cells. The MSCs secretome can be modified and improved in vitro to stimulate specific cellular effects desired for therapeutic applications, and one way of modifying it is through modifications in the culture conditions. In the present study, in addition to describing for the first time the secretome of mesenchymal stem cells derived from adipose tissue (AD-MSCs) in feline species, we identified and compared the effects exerted by different culture modifications, such as the use of serum-free medium and hypoxia in the protein production by AD-MSCs in canines and felines. For the conditioned media production, the cells were thawed and cultured with DMEM/F12 medium with 20% fetal bovine serum (FBS), supplemented with antibiotics and antimycotics in controlled incubator ay 37.5 °C with 5% CO2. After reaching at least 70% confluency, the flasks were washed three times with Hanks balanced salt solution (HBSS) and were grown in four different conditions. Conditioned media were collected after 4 days, centrifuged for 10 min/300 g, filtered on a 0.22 μm filter and frozen at -80 °C. Protein extraction... (Complete abstract click electronic access below) / Mestre

Current therapy for chronic kidney disease (CKD) is limited; however, recent studies have shown that a subpopulation of cells derived from the bone marrow, known as early outgrowth cells (EOCs), are able to attenuate kidney injury. Here we examined the efficacy of a modular tissue engineering system whereby the EOCs might be easily removed in the event of malignant change. While modular therapy mimicked the effects seen with standard EOC therapy, the modules degraded allowing the encapsulated EOCs to enter systemic circulation. Given the presumed egress of EOCs, we explored an alternative strategy for kidney protection. Here we investigated the long-term effectiveness of administering the conditioned medium (EOC-CM) that contains the factors the EOCs secrete, rather than the cells themselves. In these studies, repeated administration of EOC-CM attenuated the structural and functional manifestations of kidney injury suggesting that this approach may provide an effective and feasible, cell-free approach for CKD.

Current therapy for chronic kidney disease (CKD) is limited; however, recent studies have shown that a subpopulation of cells derived from the bone marrow, known as early outgrowth cells (EOCs), are able to attenuate kidney injury. Here we examined the efficacy of a modular tissue engineering system whereby the EOCs might be easily removed in the event of malignant change. While modular therapy mimicked the effects seen with standard EOC therapy, the modules degraded allowing the encapsulated EOCs to enter systemic circulation. Given the presumed egress of EOCs, we explored an alternative strategy for kidney protection. Here we investigated the long-term effectiveness of administering the conditioned medium (EOC-CM) that contains the factors the EOCs secrete, rather than the cells themselves. In these studies, repeated administration of EOC-CM attenuated the structural and functional manifestations of kidney injury suggesting that this approach may provide an effective and feasible, cell-free approach for CKD.

Le cartilage articulaire est un tissu possédant une faible capacité de réparation intrinsèque. Dès lors, la répétition de traumatismes articulaires induit un microenvironnement propice à la dégradation du cartilage et, in fine, l’émergence de l’arthrose. Les traitements utilisés à l’heure actuelle visent uniquement à soulager la douleur, réduire l’inflammation et la progression de l’arthrose. Ainsi, le traitement des lésions chondrales équines revêt une importance majeure puisque les affections locomotrices constituent la première cause de baisse de performances et d’arrêt prématuré de la carrière du cheval sportif. De plus, le cheval est le modèle animal qui possède le cartilage articulaire le plus semblable à celui de l’Homme et, en conséquence, représente un modèle d’étude pertinent pour les lésions chondrales humaines. Dans ce contexte, notre étude s’est attachée à développer de nouvelles stratégies pour le traitement des lésions chondrales basées sur la différenciation chondrogénique de CSM en vue de produire in vitro un substitut cartilagineux implantable en site articulaire. Ainsi, nous avons d’abord isolé et caractérisé des CSM équines à partir de prélèvements de moelle osseuse (MO) et de sang de cordon ombilical (SCO), puis, nous avons réalisé la différenciation en cultivant les CSM durant 14 jours en hypoxie ou normoxie au sein d’un biomatériau (éponges de collagène de types I/III), en présence de BMP-2 et TGF-β1 et de siRNA ciblant le collagène de type I et HtrA1, molécules atypiques du cartilage hyalin. Bien que ce protocole nous ait permis d’obtenir un substitut cartilagineux riche en marqueurs du cartilage hyalin comme le collagène de type II et l’agrécane, la présence du collagène de type I persistait. Nous avons donc tenté d’optimiser le protocole en allongeant le temps de culture, en utilisant le TGF-β3, et en modifiant la stratégie d’interférence par l’ARN. Cette étape nous a permis de conclure sur l’effet bénéfique de l’allongement de la culture à 28 jours et l’efficacité des facteurs chondrogéniques initialement utilisés. Néanmoins, la stratégie d’interférence par l’ARN demeure encore perfectible. Finalement, nous avons comparé la qualité du substitut cartilagineux obtenu après différenciation en fonction de la source de CSM utilisée. Les CSM de MO semblent les plus adaptées mais le protocole que nous avons utilisé n’est probablement pas le plus efficace pour induire la différenciation des CSM de SCO. Dans une partie complémentaire, bien que ces résultats soient préliminaires, nous avons montré que le sécrétome des CSM pourrait être un formidable outil afin d’améliorer le traitement des lésions chondrales. Dans leur ensemble, les résultats obtenus permettent d’avoir un regard optimiste concernant la mise en place de thérapies cellulaire et tissulaire du cartilage, aussi bien en médecine équine qu’humaine. / Articular cartilage is a tissue with low intrinsic repair abilities. Therefore, repeated traumas lead to cartilage degradation and ultimately, to the emergence of osteoarthritis (OA). Current therapies aim to reduce pain, inflammation and to prevent the progression of OA. Thus, treatment of equine chondral lesions is of major importance since locomotor disorders are the main causes of poor performance and early retirement of the athlete horses. In addition, the horse is an animal model with the most human-like articular cartilage and, therefore, represents the best relevant model to study human chondral lesions and arthropathies. In this context, our study focused on developing new strategies for the treatment of chondral lesions based on the chondrogenic differentiation of equine mesenchymal stem cells (MSC) in order to produce an in vitro neo-synthetized cartilaginous substitute, which could be implantable in the chondral lesion site. Thus, we first isolated and characterized equine MSC derived from bone marrow (BM) and umbilical cord blood (UCB). Then, we have differentiated MSC by culturing them for 14 days in hypoxia or normoxia, in a biomaterial (types I / III collagen sponges), in the presence of BMP-2 and TGF-β1 and siRNA targeting type I collagen and HtrA1, two atypical hyaline cartilage molecules overexpressed in OA. Although this protocol allowed us to obtain a cartilaginous substitute composed of large amounts of hyaline cartilage markers such as type II collagen and aggrecan, the presence of type I collagen persisted. We therefore tried to optimize the protocol by extending the culture time, using TGF-β3, and modifying the RNA-interference strategy. We have concluded on the beneficial effect of the lengthening of the culture to 28 days and the effectiveness of the chondrogenic factors initially used. Nevertheless, the RNA-interference strategy still remains perfectible. Finally, we compared the quality of the neo-synthetized cartilaginous substitute according to the source of MSC used. BM-MSC seem to be the most suitable, but the protocol we used is probably not the most effective for inducing UCB-MSC differentiation. In a complementary part, although these results are very preliminary, we have shown that the MSC secretome could be a tremendous tool to improve current therapies of chondral lesions. Overall, the results obtained make it possible to look ahead with optimism, in order to obtain future efficient cartilage tissue engineering therapies, both in equine and human medicines.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12831号 / 論医博第2080号 / 新制||医||1005(附属図書館) / 31369 / (主査)教授 川口 義弥, 教授 柳田 素子, 教授 小川 修 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium. Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex. / QC 20101125