O câncer representa desequilíbrio na homeostase do organismo e os mecanismos de defesa para controlar as células tumorais envolvem respostas do sistema imune inato e adaptativo, sendo que a primeira reação do organismo é a inflamação. O objetivo deste estudo foi caracterizar a resposta inflamatória em linhagens de camundongos C57BL/6, CD4KO, CD8KO, RAG e NUDE. Os animais foram tratados com o carcinógeno DMBA e o agente de promoção tumoral TPA. Os animais foram acompanhados e avaliados por 100 dias, após este período a pele tratada foi retirada e processada para análise de citocinas pró- e anti-inflamatórias e enzima mieloperoxidase (MPO). As citocinas pró e anti-inflamatórias e a enzima MPO foram maiores nos animais CD4KO quando comparados aos outros grupos de animais. As linhagens C57BL/6 WT, CD4KO e NUDE foram avaliadas 48 horas após tratamento com DMBA. Os animais CD4KO apresentaram maior número de neutrófilos e citocinas pró-inflamatórias quando comparados aos grupos C57BL/6 WT e NUDE, enquanto que as citocinas anti-inflamatórias não mostraram diferenças nos 3 grupos tratados. Estes resultados sugerem que os linfócitos TCD4 participam do controle da inflamação causada pelo DMBA e TPA. / Cancer represents imbalance in homeostasis and defense mechanisms to control the tumor cells involving the innate and adaptive immune responses, and the first reaction of the body is inflammation. The aim of this study was to characterize the inflammatory response in the following strains: C57BL/6 WT, CD4KO, CD8KO, RAG and NUDE mice. We treated the animals with the carcinogen DMBA as well as with TPA tumor promoting agent. The animals were followed for 100 days and evaluated after this period, the treated skin was removed and processed for analysis of pro-and anti-inflammatory and enzyme myeloperoxidase (MPO). The pro-and anti-inflammatory cytokines and MPO enzyme were higher in animals CD4KO than the other groups of animals. The C57BL/6 WT and CD4KO NUDE lines were evaluated 48 hours after treatment with DMBA. The CD4KO animals had greater numbers of neutrophils and proinflammatory cytokine than the C57BL/6 WT and NUDE mice, while anti-inflammatory cytokines showed no differences in the three treated groups. These results suggest that CD4 + T lymphocytes participate in the control of the induced by DMBA and TPA.

Introdução: Baixas concentrações séricas de hidroxivitamina D (25[OH]D) e o excesso de peso atingiram níveis epidêmicos em todo o mundo. Estudos relatam que concentrações séricas de vitamina D estão associadas às alterações lipídicas, glicolíticas e inflamatórias; e estas alterações são conhecidamente mediadas pela adiposidade. Dessa forma, a vitamina D pode atuar de forma benéfica sobre o perfil metabólico em adolescentes, adultos e idosos. Objetivo: Investigar e descrever as associações entre as concentrações séricas de 25(OH)D e o perfil metabólico, mediadas pela adiposidade em adolescentes, adultos e idosos. Metodologia: Inicialmente, foi utilizada subamostra do Inquérito de Saúde de São Paulo (ISA-Capital), estudo transversal, de base populacional (n=281), para investigar a associação entre as concentrações séricas de vitamina D e marcadores inflamatórios em adultos brasileiros. Posteriormente, foram utilizados dados do estudo Healthy Lifestyle in Europe by Nutrition in Adolescents-(HELENA), estudo multicêntrico transversal da população de adolescentes européia, com o intuito de avaliar as alterações nos marcadores lipídicos e de homeostase da glicose mediados pela deficiência de vitamina D e obesidade. Finalmente, foi analisada a amostra do estudo PHYSMED, um estudo transversal com idosos não institucionalizados para verificar associações entre concentrações séricas de vitamina D, perfil lipídico e composição corporal em idosos espanhóis aparentemente saudáveis. Resultados: Nos adultos, observou-se uma associação negativa entre as concentrações de TNF-alfa e de IL-6 e as concentrações séricas de 25(OH)D em indivíduos com peso normal. Nos adolescentes, as concentrações de 25(OH)D foram associadas de forma independente e positiva com o Quantitative Insulin Sensitivity Check Index-QUICKI (p <0.001) e negativamente associada com o IMC (p <0.05). Também foi observado que o aumento do IMC esteve associado com um aumento de 1.93 vezes maior chance de deficiência de vitamina D (IC de 95 por cento = 1.03 - 3.62; p = 0.040). Em idosos, verificou-se que as concentrações séricas de 25(OH)D foram associadas com o IMC (p = 0.04), a circunferência da cintura (p = 0.004), CT/HDL-c (p = 0.026) e o HDL-c (p = 0.001). Adicionalmente, foi observado que idosos com concentrações de HDL-c <40mg/dl possuíam 1.7 vezes maior chance de apresentarem deficiência de vitamina D em comparação com aqueles que possuíam concentrações de HDL-c >40 mg/dl (95 por cento IC = 1.10 a 2.85; p = 0.017) e o aumento na circunferência da cintura também foi associado com um maior risco de deficiência de vitamina D (95 por cento IC =0.96-1.00; p = 0.04). Conclusão: A composição corporal interage com as concentrações de 25(OH)D modulando a resposta inflamatória, à homeostase da glicose e também o perfil lipídico. Indivíduos sem deficiência de vitamina D apresentam melhor perfil metabólico e também melhor composição, sugerindo que a suficiência de vitamina D pode ter um papel importante nas condições metabólicas mediadas pela adiposidade. / Introduction: Low serum of hydroxyvitamin D (25 [OH] D) and excess weight reached epidemic levels in worldwide. Studies have reported that vitamin D serum concentrations are associated with lipid, glycolytic and inflammatory alterations; and these alterations are known to be mediated by adiposity. Thus, vitamin D may have a benefic action on the metabolic profile in adolescents, adults and elderly. Objective: To investigate and describe the associations between 25(OH)D concentrations and the metabolic profile mediated by adiposity in adolescents, adults and elderly. Methods: Initially, was used a subsample from the Health Survey of São Paulo (HS-SP), cross-sectional, population-based study (n = 281), to investigate the association between vitamin D concentrations and inflammatory biomarkers in Brazilian adults. Later, was used data from Healthy Lifestyle in Europe by Nutrition in Adolescents study - (HELENA), cross-sectional and multicenter study of the European adolescents, in order to evaluate the alterations in lipid markers and glucose homeostasis mediated by vitamin D deficiency and obesity. Finally, was analyzed the sample from PHYSMED study, a cross-sectional study with non-institutionalized elderly, to examine associations between vitamin D concentration, lipid profile and body composition in apparently healthy elderly Spanish. Results: In adults, a negative association was observed between the concentrations of TNF-alpha and IL-6 and serum 25(OH) D in normal weight subjects. In adolescents, the 25(OH) D concentration was associated positive and independently with QUICKI (p <0.001) and negatively associated with BMI (p <0.05). It was also observed that increasing BMI was associated with an increase of 1.93 times odds of vitamin D deficiency (95 per cent CI = 1.3 - 3.62; p = 0.040). In the elderly, it was found that serum of 25(OH) D was associated with the BMI (p = 00:04), waist circumference (p = 0.004), TC/HDL-c ratio (p = 0.026) and HDL -c (p = 0.001). Additionally, it was observed that elderly patients with HDL-c <40mg/dl had 1.7 times odds to develop vitamin D deficiency compared to those had concentrations of HDL-c> 40 mg / dl (95 per cent CI = 1.10 to 2.85 ; p = 0.017) and increases in waist circumference was also associated with an increased risk of vitamin D deficiency (95 per cent CI = 0.96-1.00; P = 0.04). Conclusion: Body composition interacts with 25(OH) D concentrations modulating the inflammatory response, glucose homeostasis and also the lipid profile. Individuals without vitamin D deficiency have better metabolic profile and better body composition, suggesting that vitamin D sufficiency may have an important role in the metabolic conditions mediated by adiposity.

Abstract Cardiac surgery with cardiopulmonary bypass induces the activation of systemic inflammatory response syndrome (SIRS) and results in at least some degree of global myocardial ischemia. Although these responses are usually short-lived, they may lead to serious complications and organ system failures. The present study evaluated the effects of high-dose glucose-insulin (1IU/kg/h) treatment (GIK) administered with the hyperinsulinemic normoglycemic clamp technique and a leukocyte-depleting filter on markers of systemic inflammatory response and myocardial ischemia-reperfusion injury in certain cardiac surgical risk groups. The study involved four prospective randomized controlled clinical trials and 119 patients. Cardioprotective effects were measured as myocardial enzyme release, recovery of contractile function and incidence of arrhythmias in all studies. The hemodynamic and metabolic effects of high-dose glucose-insulin treatment were evaluated in patients admitted for combined aortic valve (AS) and coronary surgery (40) and for urgent coronary surgery (39), and the latter study also involved proinflammatory cytokine and C-reactive protein analyses. The impacts of leukocyte filter on the expression of neutrophil adhesion molecules along with proinflammatory cytokines were evaluated in patients admitted for combined aortic valve (AS) and coronary surgery (20) and for solitary coronary surgery (20). The high-dose glucose-insulin treatment was associated with better preserved myocardial contractile function and less need for inotropic support after combined aortic valve and coronary surgery (I) and attenuation of postoperative CRP release after urgent coronary surgery (II). No effects on postoperative myocardial enzyme release (I, II) or on proinflammatory cytokine responses (II) were detected. The number of hypoglycemic events was low. The use of a leukocyte filter throughout the cardiopulmonary bypass period increased the neutrophil adhesion molecule CD11b expression in patients with both normal and prolonged CPB times and was associated with an enhanced proinflammatory cytokine response (III, IV). In conclusion, high-dose glucose-insulin treatment is safe, but requires strict control of blood glucose level. It reduces the need for inotropic support in patients with compromised cardiac status. The use of leukocyte filter leads to increased leukocyte activation and proinflammatory reaction.

We previously reported that Prunella vulgaris aqueous extract (PVAE) promotes hepatic glycogen synthesis and decreases postprandial hyperglycemia in ICR mice. Inflammatory cytokines play a critical role in the pathogenesis of diabetes. This study was designed to examine whether PVAE has a protective effect on IL-1β-induced apoptosis in INS-1 cells. INS-1 pancreatic β cells were plated at 2×10 6/ml and treated with PVAE (100 μg/ml) 30 min before the cells were challenged with IL-1β (10 ng/ml). Untreated INS-1 cells served as control. INS-1 cell cytotoxicity was examined by MTT and lactate dehydrogenase (LDH) activity assays. Caspase-3 activity and activation of the apoptotic signaling pathway were analyzed by western blotting. NF-κB binding activity was examined by EMSA. The levels of inflammatory cytokines in the supernatant were measured by ELISA. IL-1β treatment significantly induced INS-1 cell death by 49.2%, increased LDH activity by 1.5-fold and caspase-3 activity by 7.6-fold, respectively, compared with control cells. However, PVAE administration significantly prevented IL-1β-increased INS-1 cell death and LDH activity and attenuated IL-1β-increased caspase-3 activity. Western blot data showed that PVAE also significantly attenuated IL-1β-increased Fas, FasL and phospho-JNK levels in the INS-1 cells. In addition, PVAE treatment significantly attenuated IL-1β-increased NF-κB binding activity and prevented IL-1β-increased TNF-α and IL-6 expression in INS-1 cells. Our data suggest that PVAE has a protective effect on IL-1β-induced INS-1 cell apoptosis. PVAE also attenuates IL-1β-increased NF-κB binding activity and inflammatory cytokine expression in INS-1 cells. PVAE may have a benefit for type I diabetic patients.

The innate immune response is involved in the pathophysiology of cerebral ischemia-reperfusion (I/R) injury. Recent evidence suggests that scavenger receptors have a role in the induction of innate immunity. In this study, we examined the role of scavenger receptor A (SR-A) in focal cerebral I/R injury. Both SR-A-/- mice (n=10) and age-matched wild-type (WT) mice (n=9) were subjected to focal cerebral ischemia (60 minutes), followed by reperfusion (for 24 hours). Infarct size was determined by TTC (triphenyltetrazolium chloride) staining. The morphology of neurons in the brain sections was examined by Nissl's staining. Activation of intracellular signaling was analyzed by western blot. Cerebral infarct size in SR-A -/- mice was significantly reduced by 63.9% compared with WT mice after cerebral I/R. In SR-A -/- mice, there was less neuronal damage in the hippocampus compared with WT mice. Levels of FasL, Fas, FADD, caspase-3 activity, and terminal deoynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling-positive apoptotic cells were significantly increased in WT mice after cerebral I/R, but not in SR-A -/- mice. Cerebral I/R increased nuclear factor-B activation in WT mice, but not in SR-A -/- mice. These data suggest that SR-A has a central role in cerebral I/R injury and that suppression of SR-A may be a useful approach for ameliorating brain injury in stroke patients.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20690号 / 工博第4387号 / 新制||工||1682(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 高野 裕久, 教授 米田 稔, 准教授 上田 佳代 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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No description available.

There were three objectives of this study. The first was to determine if commercially available fluorochromes could be used to determine the glucocorticoid receptor (GR) density and binding affinity (BA) in equine peripheral blood mononuclear cells. The second was to determine if there was a correlation between elevated plasma cortisol and GR density or binding affinity in healthy adult horses. The third objective was to evaluate the HPA axis in adult horses presenting with systemic inflammatory response syndrome (SIRS), and to determine where any alterations in HPA axis function occur in these patients compared to healthy adults. For the first part of the study, peripheral venous blood was collected from 3 healthy research horses on 3 days. Peripheral blood mononuclear cells were isolated using Ficoll gradient centrifugation. Phycoerythrin (PE)-CD44 was then used to extracellularly label leukocytes, and then an intracellular GR antibody was used to determine a baseline measurement of GR density and fluorescein isothiocyanate (FITC)-dexamethasone was used to determine binding affinity via flow cytometric analysis. Comparison of control samples to those for CD44, GR density, and GR binding affinity showed a statistically significant difference for all samples (P<0.0001, P<0.0001, and P<0.0001 respectively). This showed that the CD44, GR antibody, and FITC-dexamethasone could successfully be used to analyze equine peripheral blood mononuclear cells for GR activity. For the second part of the study, an ACTH stimulation test was performed on 8 healthy horses in order to induce an increase in endogenous cortisol production. Plasma cortisol levels, GR density, and GR binding affinity were measured at baseline, 4, 8, and 24 hours after treatment. Median basal cortisol concentration was 4.9, range 3.2-6.1 μg/dl. This initially increased following ACTH stimulation to 5.6, range 4.8-7.4 μg/dl, then showed a significant decrease by 8 hours post ACTH administration to 1.4, range 1.1-2.7 μg/dl (P=0.0221). No correlation was observed between plasma cortisol concentration in healthy horses and GR density or binding affinity (r=-0.145, P=0.428 and r=0.046, P=0.802, respectively). For the third phase of the study, horses (N=10) with systemic inflammatory response syndrome (SIRS) were compared to healthy, age and sex matched controls (N=10) presenting for lameness evaluation or ophthalmologic examination. Blood was collected from SIRS cases and controls on presentation to the Equine Medical Center. A CBC, serum biochemistry, and serum ACTH and cortisol measurements were performed. GR density and binding affinity were also determined. Nonsurvivors had a significantly decreased GR binding affinity (P=0.008) and demonstrated a trend towards an increase in the ACTH:cortisol ratio. ROC analysis was performed for serum ACTH and cortisol concentrations, the ACTH:cortisol ratio, GR density and GR binding affinity, and triglycerides to determine cut-off values associated with nonsurvival. These were then used to analyze this population using Fischer's exact test to determine the odds ratio (OR) associated with nonsurvival for each variable. This revealed that a serum triglyceride concentration greater than 28.5 mg/dl was associated with nonsurvival (OR=117, 95% CI, 1.94-7060). The other variables were not found to be significantly associated with nonsurvival, although a Delta BA% of less than 35.79% was found to be closely associated with nonsurvival (OR=30.33, 95% CI, 0.96-960.5). Additionally, a significant negative correlation was detected between the plasma ACTH concentration and Delta BA% (r=-0.685, P=0.029) and the ACTH:cortisol ratio and the Delta BA% (r=-0.697, P=0.025). This study showed that nonsurviving horses with SIRS had a significantly decreased GR binding affinity compared to survivors, and a tendency toward an increase in their ACTH:cortisol ratios. This confirms that HPA axis dysfunction occurs in adult horses with SIRS as tissue resistance to glucocorticoids, and potentially relative adrenal insufficiency as well. These results suggest that there are horses with SIRS that might benefit from "physiologic" doses of synthetic glucocorticoids to complement their relative adrenal insufficiency in addition to their poor tissue sensitivity. Further research should focus on methods to more rapidly determine which horses might benefit from treatment with glucocorticoids on presentation, as well as to more accurately determine prognosis for survival. / Master of Science

Maturity-onset diabetes of the young (MODY), due to hepatocyte nuclear factor 1 alpha mutations (HNF1A-MODY), is the most common form of monogenic diabetes presenting in young adults. An accurate genetic diagnosis of HNF1A-MODY has therapeutic implications for the patients and their family members. However, the majority of people with HNF1A-MODY are not referred for genetic testing and remain misdiagnosed as type 1 or type 2 diabetes. As part of measures to address this misdiagnosis, over the last few years there have been efforts to define clinical features and biomarkers that can be used to identify those at high risk of HNF1A-MODY. Secreted hepatic proteins regulated by HNF1A are attractive candidates for diagnostic biomarkers that would be specific for this form of diabetes. Apolipoprotein M (apoM), C-reactive protein (CRP) and plasma glycan profile have all been investigated as biomarkers to improve selection of suspected MODY cases for genetic testing. In my thesis, I have addressed questions about the variation in apoM between different forms of diabetes and assessed the performance of hsCRP and plasma glycan profile to identify HNF1A-MODY in previously uninvestigated individuals with young-onset diabetes and in a non-European population. Additionally because CRP and plasma glycans are both important components of an acute inflammatory response, I examined the effect of haploinsufficiency of HNF1A in a standardised model of inflammation. When investigating apoM, I showed that serum apoM levels are lower in HNF1A-MODY than controls, and have demonstrated for the first time that serum apoM provides good discrimination between HNF1A-MODY and type 1 diabetes. CRP and plasma glycan profile both performed well in identifying HNF1A-MODY cases in unselected young adults with diabetes. The results also suggested that both biomarkers have value for assessing the functional impact of novel HNF1A variants. I went on to examine the use of a low CRP for selecting those at risk of HNF1A-MODY in South Asian subjects with young-onset diabetes. This study suggests that the overall population prevalence of HNF1A-MODY is similar in South Asians to Europeans, but that MODY represents a lower proportion of those with diabetes (due to the higher prevalence of type 2 diabetes in South Asians). The specific selection strategy employed in this study was not successful in identifying subjects at high risk of HNF1A-MODY (only 3% of those sequenced had mutations), suggesting that additional clinical and biochemical features will be required in addition to CRP to distinguish South Asians at high risk of HNF1A-MODY. Lastly, using endotoxaemia as a standardised model of acute inflammation for the first time in HNF1A-MODY, I have shown that despite low baseline levels, subjects with HNF1A-MODY had peak stimulated CRP levels comparable to non-diabetic controls. An attenuated cytokine response was observed in HNF1A-MODY, which requires further investigation. This is also the first report of inflammation-associated changes in plasma and white cell membrane glycan profile in diabetes. This research work adds substantially to current understanding of performance of HNF1A-MODY biomarkers, a critical step before their clinical translation. The work presented also provides novel insights into the regulation of the acute inflammatory response in HNF1A-MODY.