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In dieser Arbeit wurde in einem Maus Tumormodell untersucht, ob durch eine intraoperative Vakzination mit gentechnisch modifizierten autologen Tumorzellen ein antitumoraler Effekt erzielt werden kann. Das Experiment erfolgte mit zwei Tumorzelllinien (B16 Melanom und Lewis Lung Karzinom). Nach Implantation der Tumorzellen in C57/BL 6 Mäuse wurden diese chirurgisch entfernt. Intraoperativ erhielten die Mäuse eine Vakzination. Dazu wurden folgende Impfstoffe verwendet: 1. subletal bestrahlte mIL-12 transfizierte Tumorzellen, 2. subletal bestrahlte pRSC transfizierte Tumorzellen und 3. frostgeschockte Tumorzellen. Die Impfung erfolgte entweder subcutan oder direkt in die Milz. Es wurde die Hypothese aufgestellt, dass eine Injektion in die Milz und eine Modifikation mit IL-12 den besten Effekt erzielt. Eine Kontrollgruppe blieb ohne Vakzin. Beobachtet wurde das Tumorwachstum, der Zeitpunkt bis zum makroskopischen Wiederauftreten eines Tumors, Überlebenszeit und die Metastasierungsrate. Versuchstiere ohne Rezidivtumor erhielten erneut einen Tumor. Es erfolgte eine erneute Evaluation des Tumorwachstums, des Zeitpunktes bis zum makroskopischen Wiederauftreten eines Tumors, der Überlebenszeit und der Metastasierungsrate. In beiden Tumorzelllinien profitierten alle Therapiegruppen nach Tumorresektion und Vakzination bezüglich Tumorrezidivrate, Zeit bis zum makroskopischenWiederauftreten des Tumors, Überlebenszeit, Metastasierungsrate und Tumorwachstumsgeschwindigkeit gegenüber der Kontrollgruppe. Vereinzelt konnten signifikante Vorteile für die direkt in die Milz applizierte Vakzine bezüglich der Tumorwachstumsgeschwindigkeit aufgezeigt werden. Weiterhin ergab sich eine geringere Tumorrezidivrate, wenn IL-12 modifizierte autologe Tumorzellen nach R0 Resektion direkt in die Milz appliziert wurden. Auch nach Tumorreimplantation konnte bezüglich Überlebenszeit und Tumorwachstumsgeschwindigkeit ein Vorteil für alle Therapiegruppen gegenüber der Kontrollgruppe herausgearbeitet werden. Nach Impfung in die Milz zeigte sich tendenziell eine geringere Metastasierungsrate. Intraoperative autologe Tumorzellvakzinationen konnten im Tiermodell in einem adjuvanten Setting einen antitumoralen Effekt auslösen. Möglicherweise kann diese Art der Impfung eine zusätzlich hilfreiche Behandlungsform zu den bisherigen adjuvanten Chemotherapeutika werden.

Osteoarthritis (OA) is the most prevalent arthritis in the world with increasing numbers of people expected to acquire the disease as the population ages. Therapies commonly used to manage the disease have limited efficacy and some carry significant risks. Current data suggest that the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra) can alter the inflammatory response and cartilage erosion present in OA. Intra-articular gene expression of IL-1Ra has shown promising results in animal models to provide symptomatic improvement and minimize osteoarthritic changes. Orthogen AG (Dusseldorf, Germany) has developed a method to produce an autologous conditioned serum (ACS) rich in IL-1Ra marketed as Orthokine®. Study participants treated with ACS have improved pain and function; however, these results are preliminary and need confirmation. If ongoing trials prove that ACS can retard cartilage degeneration and reduce inflammation, the management of OA would be dramatically altered, perhaps providing a mechanism to prevent the disease or at least its progression.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18131号 / 医博第3851号 / 新制||医||1001(附属図書館) / 30989 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 坂井 義治, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Stem cell transplant is often considered the last hope for the survival for many cancer patients. The CD34+ cell content of a collection of stem cells has appeared as the most reliable indicator of the quantity of desired cells in a peripheral blood stem cell harvest and is used as a surrogate measure of the sample quality. Factors predicting the yield of CD34+ cells in a collection are not yet fully understood. Throughout the literature, there has been conflicting evidence with regards to age, gender, disease status, and prior radiation. In addition to the factors that have already been explored, we are interested in finding a cancer-chemotherapy interaction and to develop a predictive model to better identify which patients will be good candidates for this procedure. Because the amount of CD34+ cells is highly skewed, most traditional statistical methods are inappropriate without some transformation. A Bayesian generalized regression model was used to explain the variation of CD34+ collected from the sample by the cancer chemotherapy interaction. Missing data was modeled as unknown parameters to include the entire data set in the analysis. Posterior estimates are obtained using Markov chain methods. Posterior distributions identified weight and gender as well as some cancer-chemotherapy interactions as significant factors. Predictive posterior distributions can be used to identify which patients are good candidates for this procedure.

Acute myeloid leukemia (AML) is a fatal cancer of the human hematopoietic system characterized by the rapid accumulation of non-functional, immature hematopoietic cells in the bone marrow (BM) and peripheral blood (PB) of affected patients. Limited sources of safe hematopoietic stem/progenitor cells (HSPCs) for transplantation and incomplete mechanistic understandings of disease initiation, progression and maintenance have impeded advances in therapy required for improvement of long-term AML patient survival rates. Toward addressing these unmet clinical needs, the ability to generate induced pluripotent stem cells (iPSCs) from human somatic cells may provide platforms from which to develop patient-specific (autologous) cell-based therapies and disease models. However, the ability to generate iPSCs from human AML patient somatic cells had not been investigated prior to this dissertation. Accordingly, I hypothesized that cellular reprogramming of human AML patient somatic cells to iPSCs is possible and will enable derivation of autologous sources of normal and dysfunctional hematopoietic progenitor cells (HPCs). I first postulated that reprogramming AML patient fibroblasts (AML Fibs) to pluripotency would provide a novel source of normal autologous HPCs. Our findings revealed that AML patient-specific iPSCs devoid of leukemia-associated aberrations found in the matched bone marrow (BM) could be generated from AML Fibs, and demonstrated that this cellular platform allowed for the derivation of healthy HPCs capable of normal differentiation to mature myeloid lineages in vitro. During the tenure of these experiments we also redefined conventional reprogramming methods by discovering that OCT4 transcription factor delivery combined with culture in pluripotent-supportive media was minimally sufficient to induce pluripotency in AML and normal Fibs. Toward capturing and modeling the molecular heterogeneity observed across human AML samples in vitro, we next asked whether reprogramming of AML patient leukemic cells would enable generation of iPSCs and derivative HPCs that recapitulated dysfunctional differentiation features of primary disease. Our results demonstrated that conventional reprogramming conditions were insufficient to induce pluripotency in leukemic cells, but that generation of AML iPSCs could be reproducibly achieved in one AML sample when reprogramming conditions were modified. These AML iPSCs and their derivative HPCs harboured and expressed the leukemia-associated aberration found in the BM leukemic cells and similarly possessed dysfunctional differentiation capacities. Together, this body of works provides the proof of principle that cellular reprogramming can be applied on a personalized basis to generate normal and dysfunctional HPCs from AML patient somatic cells. These foundational findings should motivate additional studies aimed at developing iPSC-based cell therapies and disease models toward improving AML patient quality of life and long-term survival rates. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(社会健康医学) / 甲第23819号 / 社医博第119号 / 新制||社医||12(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 湊谷 謙司, 教授 永井 純正, 教授 波多野 悦朗 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

Yes / Introduction: Skin ageing involves senescent fibroblast accumulation, disturbance in extracellular matrix (ECM) homeostasis, and decreased collagen synthesis. Objective: to assess a cell therapy product for aged skin (RCS-01; verum) consisting of ~25 × 106 cultured, autologous cells derived from anagen hair follicle non-bulbar dermal sheath (NBDS). Methods: For each subject in the verum group, 4 areas of buttock skin were injected intradermally 1 or 3 times at monthly intervals with RCS-01, cryomedium, or needle penetration without injection; in the placebo group RCS-01 was replaced by cryomedium. The primary endpoint was assessment of local adverse event profiles. As secondary endpoints, expression of genes related to ECM homeostasis was assessed in biopsies from randomly selected volunteers in the RCS-01 group taken 4 weeks after the last injection. ­Results: Injections were well tolerated with no severe adverse events reported 1 year after the first injection. When compared with placebo-treated skin, a single treatment with RCS-01 resulted in a significant upregulation of TGFβ1, CTGF, COL1A1, COL1A2, COL3A1, and lumican mRNA expression. Limitations: The cohort size was insufficient for dose ­ranging evaluation and subgroup analyses of efficacy. Conclusions: RCS-01 therapy is well tolerated and associated with a gene expression response consistent with an improvement of ECM homeostasis. / Replicel Life Sciences Inc, Vancouver, Canada.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13636号 / 論医博第2327号 / 新制||医||1074(附属図書館) / (主査)教授 川上 浩司, 教授 森本 尚樹, 教授 安達 泰治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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