Host sensors that recognize pathogen associated molecular patterns and the mechanisms of innate immune response to mouse polyomavirus (MPyV) infection were the main topics of current work. We found that MPyV did not induce interferon (IFN) production during early events of infection, but induced interleukin-6 (IL-6) and other cytokine production without inhibiting virus multiplication. Cytokine microenvironment changed the phenotype of adjacent non infected fibroblasts toward the cancer-associated fibroblast (CAF)-like phenotype. We identified Toll-like receptor 4, a sensor of the innate immunity system, to be responsible for infection dependent IL-6 production. In an effort to determine whether and where virions are released from endosomal compartments into the cytosol, we found that the hydrophobic domains of minor capsid proteins, exposed on the surface of virions after their partial disassembly in the ER, play an important role in effective escape of virions from the lumen part of endoplasmic reticulum into the cytosol, Although naked, partially disassembled virions appear before translocation to the nucleus in the cytosol, viral DNA is not recognized by cytosolic sensors at this phase of infection Sensing of MPyV resulting in IFN production occurs first during viral replication. Mutant virus,...

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第20316号 / 薬博第826号 / 新制||薬||240(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 橋田 充, 教授 髙倉 喜信, 教授 山下 富義 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No description available.

No description available.

Toll-like receptors (TLRs) are a major group of pattern recognition receptors expressed on the surface of immune cells that recognize molecular patterns associated with all classes of pathogenic microorganisms. TLR4 recognizes the lipopolysaccharide component of Gram-negative bacterial cell walls and is the only TLR known to induce signaling through both the MyD88 and TRIF pathways. Basigin, a ubiquitous cell adhesion molecule, is a member of the immunoglobulin superfamily that has the ability to influence cell signaling mediated by the MyD88 and TRIF pathways, the same signaling pathways induced by the TLR4 receptor protein. Analysis of the Basigin protein sequence indicates the presence of a hydrophilic glutamate residue within the hydrophobic transmembrane domain, but no consensus binding sites for MyD88 or TRIF. The purpose of this study was to determine if Basigin uses TLR4 for signal transduction. It is hypothesized that Basigin interacts with TLR4 and that the glutamate residue plays a role in the interaction. Enzyme-linked immunosorbent binding assays were performed using endogenous TLR4 and recombinant Basigin proteins. These analyses demonstrated that binding of Basigin to TLR4 was significantly greater than that of the control protein and that the glutamate residue in the Basigin transmembrane domain does play a role in the interaction between Basigin and TLR4 as well as many hydrophobic residues in the Basigin transmembrane domain. The data suggest that Basigin interacts with TLR4 to influence signaling cascades using MyD88 and TRIF.

O diabetes mellitus é um grupo heterogêneo de distúrbios metabólicos caracterizado pela hiperglicemia. Indivíduos diabéticos possuem maior susceptibilidade a infecções comparado a indivíduos sadios e a hiperglicemia é um dos principais fatores que contribuem para isso, em parte, por alterar a resposta imune. Sendo assim, os macrófagos, como células essenciais para a resposta inflamatória, podem apresentar importante papel na resposta imune alterada de indivíduos diabéticos. Neste estudo, investigamos como a hiperglicemia modula os macrófagos derivados da medula óssea (BMDMs) sob um estímulo inflamatório. Para realizar este estudo, os BMDMs de camundongos C57BL/6 machos não diabéticos e diabéticos (60 mg/kg de aloxana, iv) (CEUA / FCF / USP-488) foram cultivados sob condições normais de glicose (5,5 mM) e alta concentração de glicose (25 mM ou 40 mM) e estimuladas ou não com lipopolissacarídeo (LPS, 100 ng/mL). Em comparação com os BMDMs dos camundongos não diabéticos, os BMDMs dos camundongos diabéticos estimulados com LPS apresentaram menor expressão de CD38 no tempo basal e após 24 horas, além de menor expressão de receptor do tipo Toll (TLR)-4 na superfície celular, menor capacidade fagocítica e redução na secreção de óxido nítrico, lactato, fator de necrose tumoral- e interleucina (IL)-10, porém apresentaram maior expressão de CD80, CD86 e MHC-II, maior consumo de oxigênio e maior fosforilação em quinase ativada por estresse/quinase Jun-amino-terminal (SAPK/JNK) subunidade p46 e em quinase regulada por sinal extracelular (ERK) subunidade p42, proteína quinase B (AKT) e proteína quinase C (PKC)-δ assim como maior secreção de IL-6. Quando os BMDMs dos camundongos não diabéticos foram cultivados sob condições de alta concentração de glicose in vitro e estimulados com LPS, a expressão de TLR4 e os níveis de óxido nítrico e peróxido de hidrogênio foram reduzidos. Por outro lado, os BMDMs diabéticos que também foram cultivados em alta concentração de glicose in vitro apresentaram níveis aumentados de lactato e fosforilação reduzida em AKT e PKC-δ, porém apresentaram fosforilação aumentada em p46 SAPK/JNK. A alta concentração de glicose parece modificar o comportamento dos macrófagos, afetando diferentes aspectos dos BMDMs diabéticos e não diabéticos sob estímulo de LPS, assim a hiperglicemia deixa um legado de glicose, induzindo uma memória glicêmica, alterando o estado basal dos macrófagos, modificando a via de sinalização do TLR4 contribuindo para a susceptibilidade de indivíduos diabéticos a infecções. / Diabetes mellitus is a heterogeneous group of metabolic disorders characterized by hyperglycemia. Diabetic individuals are more susceptible to infections compared to healthy subjects, and hyperglycemia is one of the major contributing factors, partly because they alter the immune response. Thus, macrophages, as essential cells for the inflammatory response, may play an important role in the altered immune response of diabetic individuals. In this study, we investigated how hyperglycemia modulates bone marrow derived macrophages (BMDMs) under an inflammatory stimulus. To perform this study, BMDMs from non-diabetic male and diabetic C57BL/6 mice (60 mg / kg aloxane, iv) (CEUA / FCF / USP-488) were cultured under normal glucose conditions (5.5 mM) and high glucose concentration (25 mM or 40 mM) and stimulated or not with lipopolysaccharide (LPS, 100 ng / ml). Compared to non-diabetic mice BMDMs, the BMDMs of LPS-stimulated diabetic mice showed lower expression of CD38 at baseline and after 24 hours, as well as lower Toll-like receptor (TLR)-4 on the cell surface, lower secretion of lactate, tumor necrosis factor-, and interleukin (IL)-10, but showed higher expression of CD80, CD86 and MHC-II, higher oxygen consumption and greater phosphorylation in stress-activated kinase/Jun-amino-terminal kinase (SAPK / JNK) p46 subunit and in extracellular signal regulated kinase (ERK) p42 subunit, protein kinase B (AKT) and protein kinase C (PKC)-δ as well as higher secretion of IL-6. When the BMDMs of nondiabetic mice were cultured under conditions of in vitro high glucose concentration and stimulated with LPS, the levels of TLR4 expression, nitric oxide and hydrogen peroxide were reduced. On the other hand, diabetic BMDMs that were also cultured in high glucose concentration of glucose in vitro showed increased levels of lactate and reduced phosphorylation in AKT and PKC-δ, but showed increased phosphorylation in p46 SAPK/JNK. A high glucose concentration seems to modify the behavior of macrophages, affecting different aspects of diabetic and non-diabetic BMDMs under the same LPS stimulus. Hyperglycemia leaves a glucose legacy, inducing a glycemic memory, altering the basal state of macrophages, modifying the TLR4 signaling pathway, and may play a key role in the high susceptibility of diabetic individuals to infections.

Swine barn air is a heterogeneous mixture of dust, bacteria and irritant chemicals including ammonia and hydrogen sulphide. Gram-negative bacteria are commonly found in swine barn air and significantly contribute to pulmonary disease in unprotected swine barn workers, through the endotoxin moiety, lipopolysaccharide (LPS). Toll-like Receptor-4 is the ligand for LPS. It is found on many cell types including monocytes, macrophages, neutrophils, endothelial cells, and to a lesser extent, epithelial cells. The severity and outcome of acute lung injury following barn air exposures depends upon the balance between epithelial and vascular endothelial repair mechanisms, including angiogenesis. Vascular Endothelial Growth Factor (VEGF) is an endothelial mitogen produced by mesenchymal and alveolar Type II epithelial cells and by activated bronchial airway epithelial cells. Research investigating the role of cytokines in angiogenesis has shown that close proximity of immune cells and endothelial cells modulates the production of various compounds that regulate vascular function. Given that LPS is the ligand for TLR4 there appeared to be a role for TLR4 in angiogenesis, particularly following endotoxin exposure. To determine whether this was occurring, we examined whether exposure to swine barn air alters vascular density in the lungs and the role of TLR4 using a murine model. Toll-like Receptor-4 wild-type (C3HeB/FeJ) and TLR4 mutant (C3H/HeJ) mice were obtained and exposed to swine barn air for 1-, 5-, or 20-days for 8 hours/day. Wild-type animals showed a 127% increase in vascular density after 20-days barn air exposure. Vascular Endothelial Growth Factor-A protein levels were decreased by 0.62-fold after one-day swine barn air exposure in wild-type animals, indicating that VEGF-A is being used as a pro-angiogenic mitogen. Transcription of VEGF-A mRNA was increased in wild-type animals after all swine barn air exposure periods. The receptor VEGFR-1 showed increased mRNA transcription over all time points. These effects were only observed in TLR4 wild-type animals, indicating that these effects are mediated by TLR4. Further, VEGF-A and VEGFR-1 appear to be involved in the manifestation of TLR4-induced angiogenesis in the lung.

Swine barn air is a heterogeneous mixture of dust, bacteria and irritant chemicals including ammonia and hydrogen sulphide. Gram-negative bacteria are commonly found in swine barn air and significantly contribute to pulmonary disease in unprotected swine barn workers, through the endotoxin moiety, lipopolysaccharide (LPS). Toll-like Receptor-4 is the ligand for LPS. It is found on many cell types including monocytes, macrophages, neutrophils, endothelial cells, and to a lesser extent, epithelial cells. The severity and outcome of acute lung injury following barn air exposures depends upon the balance between epithelial and vascular endothelial repair mechanisms, including angiogenesis. Vascular Endothelial Growth Factor (VEGF) is an endothelial mitogen produced by mesenchymal and alveolar Type II epithelial cells and by activated bronchial airway epithelial cells. Research investigating the role of cytokines in angiogenesis has shown that close proximity of immune cells and endothelial cells modulates the production of various compounds that regulate vascular function. Given that LPS is the ligand for TLR4 there appeared to be a role for TLR4 in angiogenesis, particularly following endotoxin exposure. To determine whether this was occurring, we examined whether exposure to swine barn air alters vascular density in the lungs and the role of TLR4 using a murine model. Toll-like Receptor-4 wild-type (C3HeB/FeJ) and TLR4 mutant (C3H/HeJ) mice were obtained and exposed to swine barn air for 1-, 5-, or 20-days for 8 hours/day. Wild-type animals showed a 127% increase in vascular density after 20-days barn air exposure. Vascular Endothelial Growth Factor-A protein levels were decreased by 0.62-fold after one-day swine barn air exposure in wild-type animals, indicating that VEGF-A is being used as a pro-angiogenic mitogen. Transcription of VEGF-A mRNA was increased in wild-type animals after all swine barn air exposure periods. The receptor VEGFR-1 showed increased mRNA transcription over all time points. These effects were only observed in TLR4 wild-type animals, indicating that these effects are mediated by TLR4. Further, VEGF-A and VEGFR-1 appear to be involved in the manifestation of TLR4-induced angiogenesis in the lung.

L'inflammation joue un rôle clé dans de nombreuses pathologies, telles que les maladies inflammatoires chroniques, les désordres métaboliques et le cancer. L'un de ses médiateurs le plus puissant est l'interleukine-1β (IL-1β), qui est une cytokine pro-inflammatoire participant à tous les stades de l'inflammation et de l'immunité. Son activation est régulée par un complexe multi-protéique nommé inflammasome, dont la caspase-1 active en découlant est responsable du clivage et de la maturation de l'IL-1β. Huit types d'inflammasomes activant et clivant la pro-caspase-1 ont été identifiés et contiennent tous la protéine ASC (apoptosis-associated speck-like protein containing a CARD). Les inflammasomes partagent un signal intracellulaire commun et le mécanisme menant à leur assemblage et leur activation n'est pas totalement élucidé. L'utilisation de la lignée cellulaire humaine monocytaire, THP-1, différenciée en macrophages grâce à un ester de phorbol, le TPA, a permis la mise en place d'un modèle d'étude de modulateurs de l'inflammasome en conditions stériles. Ce modèle a permis l'étude des mécanismes impliqués suite à des signaux issus de l'inflammation chronique, tels que l'ATP et les espèces réactives de l'oxygène (ROS). Ce travail montre qu'il existe une synergie entre ATP et ROS, qui agissent grâce à une boucle d'activation impliquant probablement plusieurs inflammasomes, dont NLRP3. Des donneurs de NO connus (trinitrine et isosorbide dinitrate) ou nouveau (dérivé de purine) ont montré une activité anti-inflammatoire. D'autres composés ont été identifiés comme de potentiels inhibiteurs d'inflammasome (extraits de dattes et dérivé de purine portant un acide lipoïque)

Morphine is a natural opioid which is used in medicine due to his potent analgesic and sedative effects. In the forefront of scientific interest is a chronic usage of opioids which can lead to a development of drug addiction. Morphine role in oxidative stress was described in last years. It was revealed its protective potencial by many studies. However, some studies described its pro-oxidative effect. The aim of this study was to determinate effect of chronic morphine on cell survival after oxidative stress caused by H202 analog - tBHP in the SH-SY5Y neuroblastoma cell line. The results verified morphine protective effect against oxidative stress. The highest protective effect of morphine was achieved in a concetration of 10 µM. It was desribed that morphine can induce activation of mu-opioid (MOR) and Toll-like 4 (TLR4) receptors signalling pathway on molecular level. The aim of this thesis was to evaluate the role of MOR a TLR4 in protective effect of morphine against oxidative stress by two methods. Firstly, it was used tests of oxidative stress on cell viability. The obtained results demonstrated majority role of TLR4 and minory role of MOR. Afterwards, we assesed changes in the expression of MOR a TLR4 after chronic morphine by SDS-PAGE electrophoresis. Results of these experiments did not...