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No. of bitstreams: 1 girardi_am_dr_jabo.pdf: 19183057 bytes, checksum: cf7a9853bcc87aef37b3ca91a2afceea (MD5) Previous issue date: 2016-01-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Este estudo avaliou o uso da termografia infravermelha e do exame radiológico digital como ferramentas de diagnóstico precoce da laminite em ovinos com acidose ruminal subaguda induzida experimentalmente e os efeitos da ingestão prolongada de alta proporção de alimento concentrado sobre as variáveis clínicas e laboratoriais. Foram utilizadas sete ovelhas adultas, com cânulas ruminais permanentes, que não tiveram prévio contato com alimento concentrado. Para a indução da acidose ruminal, incluiu-se ao volumoso, diariamente, 10% de alimento concentrado até atingir 80%, porcentagem mantida até completarem 19 semanas. A observação dos sinais de diarreia deu-se do quarto ao 22º dia e de laminite do quinto ao 24º dia. O fluido ruminal foi predominantemente líquido, de odor ácido e coloração amarelada. Observou-se elevação inicial nos valores da frequência cardíaca, tempo de sedimentação e flotação no fluido ruminal, plaquetas, neutrófilos segmentados, AST, FA, GGT, cálcio ionizado, magnésio, glicose, ureia, triglicérides e bilirrubinas indireta e total. No início do experimento, notou-se redução da frequência respiratória, linfocitopenia, monocitopenia, hipocalcemia, hipofosfatemia, hiponatremia, hipoproteinemia e hipocolesterolemia. A frequência de movimentos ruminais, o tempo de redução do azul de metileno, os níveis sanguíneos de creatinina, bilirrubina direta, lactato e CK foram reduzidos, enquanto as médias de temperatura, peso e escore corporal, eosinófilos, albumina, cloretos e potássio no sangue aumentaram durante todo o período de observação. Redução do pH ruminal ocorreu nos primeiros dias, a despeito de sua manutenção acima de 5,5 durante as 19 semanas de observação. Mecanismos compensatórios respiratórios e metabólicos, principalmente a acidificação da urina, mantiveram o pH sanguíneo dentro dos limites fisiológicos considerados para a espécie ovina. Dezesseis frações proteicas séricas foram separadas pela eletroforese, dentre elas oito proteínas identificadas pelos seus pesos moleculares, de funções desconhecidas. A maioria das proteínas de fase aguda e as imunoglobulinas identificadas no fracionamento eletroforético variaram durante o período de observação, sugerindo reação inflamatória. Nenhuma alteração macroscópica foi detectada à necropsia. A radiologia digital não permitiu a identificação de alterações podais decorrentes de laminite durante o período de avaliação. A termografia infravermelha detectou aumentos na temperatura da parede do casco mesmo sem a manifestação clínica de dor, indicando que esta técnica pode ser uma ferramenta útil para detectar laminite em ovinos numa fase precoce de evolução da doença. / This study evaluated the use of infrared termography and digital radiologic examination as early diagnostic tools for laminitis in sheep with experimentally induced subacute ruminal acidosis, and the effects of long-term ingestion of a high concentrate diet on clinical and laboratory variables. Seven adult ewes with permanent rumen cannula were used, which did not have any previous ingestion of concentrate feed. For the induction of ruminal acidosis, it was added to the roughage, 10% of concentrate feed until reach 80%, percentage held to the end of the 19-week experiment. Diarrhea signs were observed from fourth to 22nd day and laminitis from fifth to 24th day. Ruminal fluid was predominantly liquid, sour-smelling and yellowish. Initial increases of heart rate, sedimentation and flotation time, platelets, segmented neutrophils, AST, ALP, GGT, ionized calcium, magnesium, glucose, urea, triglycerides, indirect and total bilirubin were noted. At the beginning of the experiment, there was reduction of respiratory rate, lymphocytopenia, monocytopenia, hypocalcemia, hypophosphatemia, hyponatremia, hypoproteinemia, and hypocholesterolemia. Ruminal movement rate, methylene blue reduction time, creatinine, direct bilirubin, lactate and CK blood levels decreased, while body temperature, weight and score, eosinophils, albumin, chloride and potassium blood levels increase throughout the period. Ruminal pH reduction occurred within the first days, despite its maintenance above 5.5 during the 19 weeks. Respiratory and metabolic mechanisms, mainly urine acidification, remained the blood pH between physiologic limits for sheep. Sixteen protein fractions were separated by electrophoresis, among them eight proteins of unknown functions, identified by their molecular weights. Most acute phase proteins and the immunoglobulins identified in the electrophoretic fractionation varied throughout the observation period, suggesting inflammation. No macroscopic alteration was detected at necropsy. The digital radiology did not allow the identification of podal changes resulting from laminitis during the trial period. Infrared thermography has detected increases in the maximum temperature of the hoof wall, even without the clinical manifestation of pain, indicating that this technique can be an useful tool to detect laminitis in sheep at an early stage of the disease. / FAPESP: 2012/09111-7

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24800号 / 医博第4992号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤 貴浩, 教授 波多野 悦朗, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

C-reactive protein (CRP) is an acute phase protein produced by hepatocytes whose serum concentration increases in inflammatory conditions including cardiovascular complications. Statins that are used in the treatment of cardiovascular diseases to reduce cholesterol also lower serum CRP levels. In human hepatoma Hep3B cells, CRP is induced in response to cytokines IL-6 and IL-1β. The objective of the study was to determine the mechanism of regulation of CRP gene expression in Hep3B cells in response to cytokines and to determine the effect of statins on CRP expression. Key findings of our research were: 1. IL-1β-activated NF-κB p50/p65 acted synergistically with IL-6-activated C/EBPβ in inducing CRP transactivation through the proximal CRP promoter. 2. A NF-κB site was localized in the proximal CRP promoter centered at position -69 overlapping the known OCT-1/HNF-1/HNF-3 sites. 3. The synergy between IL-6 and IL-1β in inducing CRP gene expression was partially mediated through the NF-κB site. 4. In the absence of C/EBPβ, a complex containing C/EBPζ and RBP-Jκ was formed at the C/EBP-p50-site. 5. Overexpressed C/EBPζ repressed both (IL-6+IL-1β)-induced and C/EBPβ-induced CRP expression. 6. OCT-1 repressed (IL-6+IL-1β)-induced CRP transactivation through the proximal CRP promoter. 7. Statins reduce cytokine-induced CRP gene expression at the transcriptional level. These findings led us to conclude that: 1. CRP transcription is determined by the relative levels of various transcription factors such as C/EBPβ, C/EBPζ, NF-κB and OCT-1 and their interaction with the proximal CRP promoter. 2. Inhibition of CRP transcription by statins is not due to an anti-inflammatory effect but due to the direct effect on CRP gene expression.

No description available.

Il processo infiammatorio è un meccanismo di difesa aspecifico innato, che costituisce una risposta protettiva dell’organismo a vari tipi di insulto (infezione, danno tissutale, trauma, stress, malattie autoimmune,). Esso comporta il rilascio in circolo di mediatori pro-infiammatori (es. citochine) and anti-infiammatori (es lipossine ed alcune citochine). Le citochine pro-infiammatorie inducono effetti infiammatori (es. anoressia e febbre) e stimolano la risposta di fase acuta (APR). Invece, le lipossine e le citochine anti-infiammatorie tendono ad attenuare l’infiammazione. Gli scopi di questa ricerca erano due: distinguere i soggetti in base al grado di severità della APR dopo il parto, e dopo stimolazione intramammaria con lipopolisaccaride (LPS) in bovine da latte sottoposte a diversi stress metabolici (NaCl, BHB, EuG e IpoG). I soggetti EuG e BHB hanno mostrato una APR più severa rispetto a IpoG e NaCl. Un ulteriore scopo è stato proposto un indice composto da alcune proteine di fase acuta al fine di stimare i processi infiammatori e le conseguenze epatiche (PICE). Le bovine con PICE più basso prima del parto, avevano più alti livelli plasmatici di citochine pro-infiammatorie e lipossine prima del parto (e mostravano una APR più severa dopo il parto), anche in assenza di sintomi clinici. / Inflammation is the innate, non-specific response of the host to disturbances in his homeostasis caused by infection, tissue injury, stress, trauma, neoplastic growth, immunological disorders. It involves pro- (e.g. cytokines) and anti-inflammatory mediators (e.g. lipoxins, some cytokines). The pro-inflammatory cytokines induce inflammatory effects (e.g. anorexia, fever) and play key roles in the stimulation of acute phase response (APR). The lipoxins and anti-inflammatory cytokines tend to mitigate the inflammation. Two were the aims of this research: to investigate in dairy cows the severity of APR at calving time as well as after intramammary lypopolysaccharide (LPS) administration in cows challenged with hyperinsulinemic hypoglycemic (HypoG, n=4), hyperinsulinemic euglycemic (EuG, n=5), hyperketonemic (BHB, n=4) and control (NaCl, n=6) clamps. Plasma samples were assayed for a wide metabolic and inflammatory profile. With respect to HypoG and NaCl animals, more severe APR was observed in EuG and BHB. A further aim was the proposal of an Index, composed by several acute phase proteins, to estimate Inflammatory Processes and Hepatic Consequences (IPHC). The dairy cows with lower IPHC after calving, had higher plasma levels of pro-inflammatory cytokines and of lipoxins before calving (and showed a stronger APR after calving); this was seen also in absence of clinical symptoms.

No description available.

Alpha-tocopherol, the most abundant form of vitamin E in man, is transported in the circulation by plasma lipoproteins. It plays important roles, not only in preventing lipid peroxidation, but also in modulating several cell functions such as cell signaling and gene expression. While chylomicrons transport dietary alpha-tocopherol after intestinal absorption, LDL and HDL are the major carriers of alpha-tocopherol in fasting plasma and largely contribute to its delivery to cells and tissues. Exchanges of alpha-tocopherol occur between plasma lipoproteins. In addition, alpha-tocopherol transfers have also been observed, in both directions, between plasma lipoproteins and artificial chylomicrons such as intravenous lipid emulsion particles used in parenteral nutrition. In acute conditions, intravenous supply of vitamin E via lipid emulsions, which bypasses the intestinal tract, may offer some advantages over oral administration to rapidly increase alpha-tocopherol plasma concentration. However, many questions remain unanswered regarding kinetics and factors facilitating vitamin E exchanges between lipid emulsions and plasma lipoproteins. The first part of this work aimed at characterizing alpha-tocopherol transfers between alpha-tocopherol rich emulsion particles and plasma lipoproteins as well as the potential for plasma proteins to modulate such transfers. An in vitro model of incubation was used in which emulsion triglyceride concentration was relatively low and lipoprotein levels comparable to those commonly found in the circulation. Results indicate a high capacity for LDL and HDL to acquire extra-amounts of alpha-tocopherol by rapid mass transfers from alpha-tocopherol-rich emulsion particles. Data further shows that, at a fixed alpha-tocopherol concentration provided by emulsion particles, the limiting factor for alpha-tocopherol enrichment is not the capacity of plasma lipoproteins to accommodate extra-amounts of alpha-tocopherol but the facilitating effect of plasma proteins on alpha-tocopherol transfer, the duration of the incubation and possibly the competition between different acceptor particles. Two lipid transfer proteins, PLTP and CETP, appear to largely mediate facilitation of alpha-tocopherol transfer; however, other plasma proteins may be involved. Data further shows that alpha-tocopherol enriched LDL and HDL can readily transfer newly acquired alpha-tocopherol to cells, without any regulation by plasma proteins.<p>Short-term prophylactic vitamin E supplementation has been suggested to be beneficial in some patients in acute conditions who present reduced plasma vitamin E concentrations in association with important changes in plasma lipids and severe oxidative stress. However, it was not clear whether low plasma vitamin E concentration in critically ill patients is related to changes in the composition of plasma lipoproteins or to a decrease in the number of alpha-tocopherol carriers. In the second part of this work, two clinical studies were conducted to analyze changes of lipoprotein concentration and composition in relation to inflammatory reaction and oxidative stress in selected subgroups of critically ill patients, namely patients undergoing cardiac surgery with different procedures. Important changes in LDL and HDL lipid content were observed, some of which contrast with previous observations made in critically ill septic patients. The reduced plasma level of alpha-tocopherol measured after cardiac surgery is entirely due to a reduced number of circulating LDL and HDL particles. Data suggests that such reduced number in alpha-tocopherol carriers post-surgery may impede the delivery of alpha-tocopherol to cells in conditions of increased requirements due to oxidative stress. Avoidance of extracorporeal circulation during cardiac surgery does not reduce inflammation-related changes in plasma lipids but largely prevents oxidative stress. This data on changes occurring in plasma lipoproteins may help to better define strategies against pro-inflammatory changes or oxidative stress. If further studies would confirm a clinical benefit with evidence-based rationale, alpha-tocopherol enriched lipid emulsions may be used to guarantee a sufficient alpha-tocopherol supply in acute conditions associated with fewer alpha-tocopherol transporters and increased requirements due to high risk of oxidative tissue injury.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

C-reactive protein (CRP) is synthesized in hepatocytes. The serum concentration of CRP increases dramatically during the acute phase response. In human hepatoma Hep3B cells, maximal CRP expression occurs in cells treated with the combination of IL-6 and IL-1β. IL-6 induces transcription of the CRP gene and IL-1β synergistically enhances the effects of IL-6. We investigated the role of IL-6-activated transcription factor STAT3, also known as STAT3α, in inducing CRP expression since we identified four consensus STAT3-binding sites centered at positions - 72, - 108, - 134 and - 164 on the CRP promoter. It has been shown previously that STAT3 binds to the site at - 108 and induces CRP expression. We found that STAT3 also bound to the other three sites, and several STAT3-containing complexes were formed at each site, suggesting the presence of STAT3 isoforms and additional transcription factors in the complexes. Mutation of the STAT3 sites at - 108, - 134 or - 164 resulted in decreased CRP expression in response to IL-6 and IL-1β treatment, although the synergy between IL-6 and IL-1β was not affected by the mutations. The STAT3 site at - 72 could not be investigated employing mutagenesis. We also found that IL-6 activated two isoforms of STAT3 in Hep3B cells: STAT3α which contains both a DNA-binding domain and a transactivation domain and STAT3β which contains only the DNA-binding domain. Taken together, these findings raise the possibility that IL-6 not only induces CRP expression but also regulates the induction of CRP expression by activating STAT3 isoforms and by utilizing all four STAT3 sites.

No description available.

No description available.