京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18900号 / 医博第4011号 / 新制||医||1009(附属図書館) / 31851 / 京都大学大学院医学研究科医学専攻 / (主査)教授 影山 龍一郎, 教授 野田 亮, 教授 楠見 明弘 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13085号 / 論医博第2126号 / 新制||医||1021(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 渡邊 直樹, 教授 岩田 想, 教授 富樫 かおり / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Nano-sized particles are widely regarded as a tool to study biological events at cellular and molecular levels. However, there are only a few imaging modalities that can visualize interactions between nanoparticles and living cells. A new technique -- pulsed magneto-motive ultrasound imaging, capable of in-vivo imaging of magnetic nanoparticles at improved depth and in real-time is introduced in this study. In pulsed magneto-motive ultrasound imaging, an external high-strength pulsed magnetic field is applied to induce motion within magnetically labeled tissue and ultrasound is used to detect the induced internal tissue motion. A laboratory prototype of a pulsed magneto- motive ultrasound imaging system was built, tested and optimized through modeling and experimental studies using tissue-mimicking phantoms, ex-vivo tissue samples and in- vivo mouse tumor model. The results demonstrated a sufficient contrast between normal and iron-laden tissue labeled with ultra-small magnetic nanoparticles. Finally, further modifications and research directions are discussed which can eventually lead to development of a clinically applicable pulsed magneto-motive ultrasound imaging system. / text

Gold nanoparticles attain an intense focus in biomedical imaging applications due to their unique optical properties, facile conjugation with biomolecules, and biocompatibility. Although a considerable amount of work towards the development of gold nanoparticles has been completed, these promising contrast agents have not yet reached the clinic due to several challenges including efficient accumulation at the diseased site, sensitivity of detection in vivo, potential adverse effects, and clearance from the body. High signal-to-background ratio is required to enhance sensitivity of detection. Because near infrared (near-IR) light has the best tissue penetration, contrast agents designed to work in this range can significantly increase imaging sensitivity. Moreover, efficient targeting of the molecular biomarkers on diseased cells can decrease the required dosage, increase the site-specific accumulation, and enhance the imaging sensitivity. Molecular-specific contrast agents developed in this project use directional attachment of antibody molecules to the nanoparticle surface, enhancing the targeting efficacy. Additionally, cell-based delivery of diagnostic and therapeutic agents is gaining much interest due to the immune cells’ special access to the avascular, diseased regions. The contrast agents developed in this project enable detection of just a few cells per unit of imaging volume, enable multiplex imaging, and open up a possibility for tracking different cell populations with noninvasive photoacoustic and ultrasound imaging. Finally, the clearance of nanoparticles from the body dictates their clinical translation. The in vivo pharmacokinetics study along with the proposed in vitro model explored in this project will enable fast, reliable, and cost-efficient screening of promising agents and facilitate quick optimization of nanoparticles for their potential use in the clinic. / text

Solid organ transplantation has evolved from being an experimental procedure to a life-saving treatment for patients with end-stage organ failure. The risk of losing a transplant due to acute rejection is very low with the use of modern immunosuppressive protocols and the short-term results are impressive. However, long-term outcomes are suboptimal and transplant recipients are at increased risks for severe complications such as cancers, opportunistic infections and cardiovascular events. The previous struggle to achieve short-term survival has turned into a search for new strategies to improve patient and transplant longevity. Regulatory T cells (TRegs), a subset of T cells, occur naturally in the immune system and have the capacity to down regulate immune responses. Under normal conditions they maintain self-tolerance and prevent excessive immune activation. Functional TReg defects lead to a massive autoimmune response and are not compatible with life. Preclinical data support that TRegs can be used as a cell therapy to prevent transplant rejection, with the potential to minimize the need for traditional immunosuppression and improve the long-term outcome. This thesis aims to enhance the translation of TReg cell therapy to clinical organ transplantation. In particular, strategies for isolation and expansion of TRegs from uremic patients awaiting kidney transplantation have been assessed. A non-invasive imaging technique to study T cell products after intravenous administration was developed, for use in future clinical trials. The performance of a novel cell purification technique was investigated to potentially improve the clinical production of TRegs. The thesis demonstrates that TRegs can be isolated and expanded from uremic patients to display potent suppressive properties in vitro. The mode of isolation and expansion affect the functional characteristics, where cells purified with cytometry based techniques and expanded with mature dendritic cells were the most advantageous. T cells can be labeled using the radioactive tracer [111In]oxine with preserved viability and subsequently followed in vivo with SPECT/CT for more than 1 week after intravenous administration. The use of microfluidic switch technology offers a novel way of purifying TRegs at high speed, purity and viability, under conditions compatible with clinical use.

<p>Luminescent semiconductor nanocrystals or quantum dots (QDs) offer attractive characteristics as a new class of fluorescent probes for molecular, cellular and in vivo imaging. While traditional cadmium-containing QDs have been widely used in biomedical research, diagnostics, and drug delivery, the cytotoxicity arising from the release of Cd2+ ions caused by the degradation of the surface coating is deemed to be a shortfall of cadmium-based QDs for long-term cellular and in vivo imaging. Here we report a direct synthesis of silver-doped zinc selenide QDs in water with near-infrared tunable fluorescence emissions, coinciding with the biological window of transmission to offer high signal-to-noise for fluorescence imaging of cells and small animals. Glutathione, which carries both carboxyl and amino groups, serves as a stabilizing ligand and offers the flexibility of decorating the surface of the QDs with moieties such as proteins, peptides and DNA. The cytotoxicity of the as-synthesized QDs was evaluated on macrophage (RAW 264.7) cells and human mesenschymal stem cells using MTS cell viability assay. The results indicated that the silver- doped ZnSe QDs possess low cytotoxicity. In vivo biodistribution study shows that these bare QDs are different from conventional QDs, it traversed through systemic route and could accumulate in the stomach of nude mice. These QDs were conjugated to monoclonal CD44v6 antibody and tested with human gastric adenocarcinoma cell line (AGS). The results indicated the feasibility of modifying the surface properties of these QDs for efficient targeting applications. The QDs were also conjugated to heparin and used to formulate nanocomplexes with chitosan to encapsulate tumor necrosis factor-alpha. Quantitative imaging analysis revealed in vivo trafficking kinetics of the nanocomplexes to the lymph nodes after subcutaneous administration into nude mice. This study demonstrates the potential of incorporation of near-infrared-emitting QDs in nanocarrier drug delivery that allows in vivo trafficking of the biodistriution events and will be of greatly improve the development new drug nanocarrier formulations.</p> / Dissertation

Made available in DSpace on 2016-05-17T16:51:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-06-12. Added 1 bitstream(s) on 2016-05-17T16:54:16Z : No. of bitstreams: 1 000863700.pdf: 3931000 bytes, checksum: 1e9e80714f2702d4fc189cd6a85f0b6e (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Visceral leishmaniasis is an important parasitic disease, with worldwide distributions that affects humans and animals. There are reports of brain involvement during the infection; however, its pathogenesis has not been completely elucidated. Therefore, with this study, we aimed the evaluation of the inflammatory response in the brain and the integrity of the blood-brain barrier in naturally infected dogs, and the establishment of an experimental mice model that could allow the evaluation of the infection dynamics, as well as the localization of the parasite and the inflammatory response in vivo. Naturally infected dogs presented a specific chemokine profile in the brain, highly attractive to T-lymphocytes, besides the evident blood-brain barrier disruption, with increased albumin quota and elevated concentrations of anti-Leishmania antibodies in the cerebrospinal fluid. Using molecular techniques and state-of-the-art in vivo imaging tools, we could determine the presence of parasites in the brain of experimentally infected Balb/c mice, as well as its intracranial localization. Brain inflammation, evaluated during four months post-infection, presented two distinct phases, one acute phase in the first fourteen days, and one phase of re-inflammation after three months of infection. Altogether, the data from this study allow us to confirm that the brain is affected during visceral leishmaniasis, and reinforce the fact that the brain involvement has been neglected during visceral leishmaniasis / FAPESP: 2012/23950-1 / FAPESP: 12/10415-0 / FAPESP: 2014/03078-3

Bacillus anthracis, l'agent infectieux responsable de la maladie du charbon, est un agent pathogène majeur du risque biologique provoqué, notamment en raison de la sévérité de la forme respiratoire de la maladie. Celle-ci résulte de l'inhalation de spores dont les mécanismes de pénétration au niveau pulmonaire sont mal connus à l'heure actuelle. Cette thèse présente les apports des microscopies confocale et biphotonique à l'étude de ces mécanismes de pénétration des spores inhalées. Le modèle murin CX3CR1+/gfp, dont la sous-population CD11b+ de cellules dendritiques (DCs) exprime constitutivement la protéine de fluorescence verte (GFP), a été utilisé dans ces travaux. Une première partie présente le développement d'une méthode automatisée de discrimination des DCs parmi d'autres populations cellulaires exprimant le même fluorophore, en se basant sur le calcul d'un coefficient morphologique. Cette méthode a permis d'étudier dans un deuxième temps le comportement spécifique de la sous-population de DCs CD11b, après infection par des spores de B. anthracis. L'étude microscopique a été d'abord effectuée in situ, c'est-à-dire sur des explants pulmonaires maintenus dans des conditions favorables à la préservation de l'activité cellulaire, puis in vivo, sur des souris anesthésiées et ventilées. Le protocole d'imagerie tire profit d'une stratégie d'acquisition et de traitement a posteriori des données permettant de surmonter, sans contrainte mécanique appliquée à l'organe, les problèmes de focalisation liés aux mouvements thoraciques durant la ventilation de l'animal. Cette stratégie originale utilise un sur-échantillonnage de l'acquisition et profite du signal de seconde harmonique généré par le collagène comme référence spatiale ; elle a permis l'observation in vivo d'interactions entre DCs et macrophages au niveau pulmonaire. Ces interactions, de type synapse immunologique, sont favorisées par l'infection et présentent donc un rôle fonctionnel qui reste à définir. La formation de synapses immunologiques entre macrophages et DCs pourrait non seulement représenter un chaînon manquant à l'explication de la pénétration des spores de B. anthracis au niveau pulmonaire, mais pourrait aussi constituer un enjeu crucial dans la compréhension de la réponse immunitaire associée aux infections pulmonaires. / Bacillus anthracis, the causative agent of anthrax, is a major bioterrorism pathogen mainly because it can lead to a severe respiratory form of the disease. This form results from inhalation of spores, whose ways of entry into the lungs are not fully understood. This thesis reports the contribution of confocal and two-photon microscopy to the study of the penetration mechanisms of inhaled spores. The animal model utilized was CX3CR1+/gfp mouse, which constitutively expresses the green fluorescent protein (GFP) on CD11b+ dendritic cells (DCs). First, we present an automated method allowing discrimination of DCs among other GFP expressing cells, based on a morphologic coefficient. This method was then applied to the study of the specific behavior of CD11b DCs, after infection by B. anthracis spores. The microscopic study was first performed in situ, i.e. on explanted organs kept in conditions favorable to cell dynamics, then in vivo, i.e. on anesthetized and ventilated mice. In this case the imaging protocol profits from both acquisition and post-processing strategies, and allowed overcoming the focalization pitfalls coming from chest movements during ventilation. This novel strategy is based on an over-sampling of frame acquisition and utilizes second harmonic generation signal from alveolar collagen as a spatial reference. It led to the first ever in vivo observation of interactions between DCs and macrophages at the lung level. These immunological synapse-like structures are promoted by infection and thus display a functional role unknown until now. The formation of macrophages-DCs immunological synapses not only could represent a missing-link in figuring out the B. anthracis spore penetration mechanisms at the lung level, but more importantly could lead to a better understanding of the immune response associated with pulmonary infections.

This dissertation is author version of following the journal article. Rei Mizuno, Yuji Kamioka, Kenji Kabashima, Masamichi Imajo, Kenta Sumiyama, Eiji Nakasho, Takeshi Ito, Yoko Hamazaki, Yoshihisa Okuchi, Yoshiharu Sakai, Etsuko Kiyokawa, and Michiyuki Matsuda. "In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines" J Exp Med 2014 211:1123-1136. Published May 19, 2014, doi:10.1084/jem.20132112 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18542号 / 医博第3935号 / 新制||医||1006(附属図書館) / 31442 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 千葉 勉, 教授 長澤 丘司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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