アサリに寄生するPerkinsus属原虫２種に関する研究

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 良永 知義, 東京大学教授 金子 豊二, 東京大学准教授 伊藤 直樹, 東京大学准教授 松本 安喜, 東京大学准教授 濱崎 恒二University of Tokyo(東京大学

Long-term Remission of Hepatitis-associated Aplastic Anemia Possibly due to Immunosuppressive Therapy after Liver Transplantation

Hepatitis-associated aplastic anemia (HAAA) is an acquired bone marrow failure syndrome that develops after seronegative fulminant hepatitis. Abnormal cytotoxic T-cell activation with cytokine release is a possible pathophysiology. We present the case of a 16-month-old Japanese male who developed HAAA following living-donor liver transplantation for fulminant hepatitis. His aplastic anemia was successfully treated with immunosuppressive therapy. He had been administered tacrolimus for prophylaxis against hepatic allograft rejection. Ten years after the HAAA onset, the patient’s bone marrow was found to be slightly hypoplastic. Tacrolimus may be effective in controlling abnormal immune reactions that can cause recurrent impaired hematopoiesis

Biochemical Studies of Mitochondrial Malate: Quinone Oxidoreductase from Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC50 of 0.822 μM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites

Transcriptional profiling of Toll-like receptor 2-deficient primary murine brain cells during <i>Toxoplasma gondii</i> infection

<div><p>Background</p><p><i>Toxoplasma gondii</i> is capable of persisting in the brain, although it is efficiently eliminated by cellular immune responses in most other sites. While Toll-like receptor 2 (TLR2) reportedly plays important roles in protective immunity against the parasite, the relationship between neurological disorders induced by <i>T</i>. <i>gondii</i> infection and TLR2 function in the brain remains controversial with many unknowns. In this study, primary cultured astrocytes, microglia, neurons, and peritoneal macrophages obtained from wild-type and TLR2-deficient mice were exposed to <i>T</i>. <i>gondii</i> tachyzoites. To characterize TLR2-dependent functional pathways activated in response to <i>T</i>. <i>gondii</i> infection, gene expression of different cell types was profiled by RNA sequencing.</p><p>Results</p><p>During <i>T</i>. <i>gondii</i> infection, a total of 611, 777, 385, and 1105 genes were upregulated in astrocytes, microglia, neurons, and macrophages, respectively, while 163, 1207, 158, and 1274 genes were downregulated, respectively, in a TLR2-dependent manner. Overrepresented Gene Ontology (GO) terms for TLR2-dependently upregulated genes were associated with immune and stress responses in astrocytes, immune responses and developmental processes in microglia, metabolic processes and immune responses in neurons, and metabolic processes and gene expression in macrophages. Overrepresented GO terms for downregulated genes included ion transport and behavior in astrocytes, cell cycle and cell division in microglia, metabolic processes in neurons, and response to stimulus, signaling and cell motility in macrophages.</p><p>Conclusions</p><p>To our knowledge, this is the first transcriptomic study of TLR2 function across different cell types during <i>T</i>. <i>gondii</i> infection. Results of RNA-sequencing demonstrated roles for TLR2 varied by cell type during <i>T</i>. <i>gondii</i> infection. Our findings facilitate understanding of the detailed relationship between TLR2 and <i>T</i>. <i>gondii</i> infection, and elucidate mechanisms underlying neurological changes during infection.</p></div

Transcriptomic Analysis of the Effects of Chemokine Receptor CXCR3 Deficiency on Immune Responses in the Mouse Brain during <i>Toxoplasma gondii</i> Infection

The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals, including humans. We previously revealed through a whole-brain transcriptome analysis that infection with T. gondii in mice causes immune response-associated genes to be upregulated, for instance, chemokines and chemokine receptors such as CXC chemokine receptor 3 (CXCR3) and its ligand CXC chemokine ligand 10 (CXCL10). Here, we describe the effect of CXCR3 on responses against T. gondii infection in the mouse brain. In vivo assays using CXCR3-deficient mice showed that the absence of CXCR3 delayed the normal recovery of body weight and increased the brain parasite burden, suggesting that CXCR3 plays a role in the control of pathology in the brain, the site where chronic infection occurs. Therefore, to further analyze the function of CXCR3 in the brain, we profiled the gene expression patterns of primary astrocytes and microglia by RNA sequencing and subsequent analyses. CXCR3 deficiency impaired the normal upregulation of immune-related genes during T. gondii infection, in astrocytes and microglia alike. Collectively, our results suggest that the immune-related genes upregulated by CXCR3 perform a particular role in controlling pathology when the host is chronically infected with T. gondii in the brain

Production of cytokines and PGE₂ by astrocytes, microglia, and peritoneal macrophages after <i>T</i>. <i>gondii</i> infection.

<p>Primary astrocytes (A), microglia (B), and peritoneal macrophages (C) from wild-type (white) and <i>Tlr2</i>^-/- mice (black) were infected with <i>T</i>. <i>gondii</i> tachyzoites. Each bar represents the mean ± SD of triplicate wells for each group. This is a representative result of two independent experiments. Asterisks represent significant differences with <i>p</i> < 0.05 in Student’s t-test.</p