Le rôle de l'inflammation dans le développement et la progression des maladies rénales chroniques (MRC) chez le chat a été peu étudié. L'hepcidine est une protéine de la phase aigue (PPA) de l'inflammation qui contribue au développement des anémies lors de MRC chez l'homme. Les objectifs de cette étude sont de comparer les concentrations en PPAs, en erythropoietine (EPO) ainsi que le statut en fer entre un groupe de chats sains et en MRC. 18 chats sains et 38 chats en MRC ont été recrutés de façon prospective. Les examens réalisés incluaient hématologie, biochimie, analyse d'urine, Serum amyloid A (SAA), haptoglobine (HAP), EPO, hepcidine,fer, TIBC et ferritinne. Nous avons observé une augmentation significative des concentrations en SAA et en hepcidine ainsi qu'une diminution significative du fer et du TIBC dans le groupe MRC (P < .05). Une corrélation positive entre la créatinine et certaines PPAs (SAA and hepcidin; P < .05) était présente. L'augmentation de SAA et hepcidine était significativement associé avec une diminution du TIBC et de l'hématocrite dans le groupe MRC. Les 14 (37%) chats anémiques du groupe MRC avaient une concentration significativement plus basse en fer et en TIBC (P < .05), changements compatibles avec une déficience fonctionelle en fer. Aucun chat n'avait un panel de fer compatible avec une carence en fer absolue. En conclusion, les résultats de cette étude suggèrent que les MRC chez le chat sont des conditions pro-inflammatoires, ayant un impact sur le métabolisme du fer. / The role of inflammation in the development and progression of feline chronic kidney disease (CKD) is not well characterized. Hepcidin, a recently discovered acute phase protein (APP) contributes to the development of anemia in human patients with CKD. The objectives of our study was to compare plasma APP including hepcidin, iron status, and erythropoietin (EPO) concentrations between healthy and cats with naturally occurring CKD. Eighteen healthy control cats and 38 cats with CKD were prospectively recruited. Complete physical examination along with hematology, biochemistry, plasma amyloid A (SAA), haptoglobine (HAP), EPO, iron, TIBC and ferritin were performed using routine laboratory analyses and commercially available feline ELISA-assays. Hepcidin-25 concentration was assessed with a human ELISA kit (DRG® Diagnostics). We found that mean SAA and hepcidin concentration were significantly higher and mean total iron and TIBC were significantly lower in the CKD group (P < .05). There was a significant positive correlation between creatinine and APPs (SAA and hepcidin; P < .05). Increase in SAA and hepcidin was also significantly associated with decrease of TIBC and PCV in the CKD group. The 14 (37%) anaemic cats with CKD had significantly and lower iron, TIBC, consistent with functional iron deficiency (P < .05). There were no patients with an iron profile suggestive of a true iron deficiency. There was no association with survival. In conclusion, our data suggest that feline CKD is a pro-inflammatory state, having significant impact on iron metabolism. With further validation, hepcidin may help better characterize these interactions.

O linfoma é uma doença neoplásica comum em cães, requerendo quimioterapia para aumentar a sobrevida dos pacientes. Durante o tratamento, são freqüentes as recidivas, que motivam alteração do protocolo medicamentoso. Proteína C-reativa e amilóide A sérica são mediadores de fase aguda produzidos no fígado que apresentam elevações de concentrações séricas em condições inflamatórias, infecciosas e neoplásicas de maneira geral. O objetivo do trabalho foi avaliar o papel dessas proteínas na monitorização da remissão e recidiva do linfoma em cães, utilizando 2 protocolos de tratamento. O protocolo COP (ciclofosfamida, vincristina e prednisona) caracterizou-se por fase de indução de 1 mês e ciclos de manutenção a cada 21 dias e o protocolo VCM (vincristina, ciclofosfamida, metotrexato e L- asparaginase) foi empregado em um regime semanal contínuo. Constituíram-se 5 grupos de estudo: Normal (20 cães hígidos), Controle COP (4 cães hígidos submetidos a quimioterapia com o protocolo COP), Controle VCM (4 cães hígidos submetidos a quimioterapia com o protocolo VCM), Linfoma COP (10 cães com linfoma multicêntrico tratados com o protocolo COP) e Linfoma VCM (10 cães com linfoma multicêntrico tratados com o protocolo VCM). Proteína C-reativa e amilóide A sérica foram determinadas pela técnica de Elisa e a eletroforese foi feita em tiras de acetato de celulose. Nos cães do grupo Normal, o estabelecimento das concentrações de proteína C-reativa, amilóide A sérica e as rações eletroforéticas ocorreu uma única vez; nos cães dos grupos Controle COP e Controle VCM na 1ª, 2ª, 3ª, 4ª, 7ª, 10ª, 13ª e 16ª semanas de quimioterapia e, nos cães dos grupos Linfoma COP e Linfoma VCM, na 1ª, 2ª, 3ª e 4ª semanas, bem como na recidiva e num momento de estabilidade da doença imediatamente antes da recidiva. Os resultados foram interpretados por análise de variância com medidas repetidas, tendo como fatores de controle os grupos e as semanas de observação, seguida de comparações múltiplas de Tukey, ao nível de significância de 5 %. Concluiu-se que: o linfoma induz a resposta de fase aguda em cães, sendo a intensidade da resposta amenizada durante o tratamento bem-sucedido dos pacientes; incrementos de proteína C-reativa e amilóide A sérica não estão relacionados à recidiva do linfoma; a quimioterapia do linfoma com os protocolos COP e VCM não altera a resposta de fase aguda, avaliada por meio dessas proteínas, nem existe diferença na resposta de fase aguda entre tais protocolos; existe relação direta entre os níveis de proteína C-reativa e amilóide A sérica no curso do linfoma e da quimioterapia; aumentos das concentrações séricas de proteína C-reativa são acompanhados de elevações da fração de &beta;2-globulinas e aumentos de amilóide A sérica são acompanhados de elevações de &beta;1-globulinas. / Lymphoma is a common neoplasm in dogs and chemotherapy is indicated to achieve long-term survivals. During the treatment, frequent relapses require drug regimen modifications. C-reactive protein (CRP) and serum amyloid A (SAA) are hepatic acute-phase mediators and usually are increased in inflammatory, infectious and neoplastic conditions. The aim of this study was to evaluate the role of these proteins in remission and relapse monitoring of dogs with lymphoma, under 2 chemotherapy protocols. COP protocol (cyclophosphamide, vincristine and prednisone) included an one-month induction period and maintenance cycles each 21 days and VCM protocol (vincristine, cyclophosphamide, methotrexate and L-asparaginase) was administered in a continuous weekly schedule. Five groups were composed: Normal (20 healthy dogs), COP Control (4 healthy dogs submitted to chemotherapy with COP protocol), VCM Control (4 healthy dogs submitted to chemotherapy with VCM protocol), COP Lymphoma (10 dogs with multicentric lymphoma treated with COP protocol) and VCM Lymphoma (10 dogs with multicentric lymphoma treated with VCM protocol). CRP and SAA were determined by Elisa tests and the electrophoresis was performed in cellulose acetate strips. In Normal dogs, CRP and SAA levels, as well the electrophoretic fractions, were measured only one time; in COP Control and VCM Control groups of dogs at the chemotherapy weeks 1, 2, 3, 4, 7, 10, 13 and 16; in dogs from groups COP Lymphoma and VCM Lymphoma, at the weeks 1, 2, 3 and 4, beside the relapse and a called stability moment immediately before the relapse. Results were compared by means of repeated measures variancy analyses, considering the groups and the observation weeks as the control factors, followed by Tukey´s multiple comparisons, at 5 % of significance level. It was concluded that: lymphoma induces an acute phase response in dogs and the intensity of response declines along the disease remission; increases of CRP and SAA are not related to lymphoma relapse; neither COP nor VCM chemotherapy changes the acute-phase response, when CRP and SAA are taken in account, and there is not difference on acute-phase response between both regimens; there is a positive correlation between CRP and SAA levels during lymphoma assessment and chemotherapy; increases of CRP levels are followed by &beta;2-globulin elevations and increases of SAA levels are related to &beta;1-globulin elevations.

O linfoma é uma doença neoplásica comum em cães, requerendo quimioterapia para aumentar a sobrevida dos pacientes. Durante o tratamento, são freqüentes as recidivas, que motivam alteração do protocolo medicamentoso. Proteína C-reativa e amilóide A sérica são mediadores de fase aguda produzidos no fígado que apresentam elevações de concentrações séricas em condições inflamatórias, infecciosas e neoplásicas de maneira geral. O objetivo do trabalho foi avaliar o papel dessas proteínas na monitorização da remissão e recidiva do linfoma em cães, utilizando 2 protocolos de tratamento. O protocolo COP (ciclofosfamida, vincristina e prednisona) caracterizou-se por fase de indução de 1 mês e ciclos de manutenção a cada 21 dias e o protocolo VCM (vincristina, ciclofosfamida, metotrexato e L- asparaginase) foi empregado em um regime semanal contínuo. Constituíram-se 5 grupos de estudo: Normal (20 cães hígidos), Controle COP (4 cães hígidos submetidos a quimioterapia com o protocolo COP), Controle VCM (4 cães hígidos submetidos a quimioterapia com o protocolo VCM), Linfoma COP (10 cães com linfoma multicêntrico tratados com o protocolo COP) e Linfoma VCM (10 cães com linfoma multicêntrico tratados com o protocolo VCM). Proteína C-reativa e amilóide A sérica foram determinadas pela técnica de Elisa e a eletroforese foi feita em tiras de acetato de celulose. Nos cães do grupo Normal, o estabelecimento das concentrações de proteína C-reativa, amilóide A sérica e as rações eletroforéticas ocorreu uma única vez; nos cães dos grupos Controle COP e Controle VCM na 1ª, 2ª, 3ª, 4ª, 7ª, 10ª, 13ª e 16ª semanas de quimioterapia e, nos cães dos grupos Linfoma COP e Linfoma VCM, na 1ª, 2ª, 3ª e 4ª semanas, bem como na recidiva e num momento de estabilidade da doença imediatamente antes da recidiva. Os resultados foram interpretados por análise de variância com medidas repetidas, tendo como fatores de controle os grupos e as semanas de observação, seguida de comparações múltiplas de Tukey, ao nível de significância de 5 %. Concluiu-se que: o linfoma induz a resposta de fase aguda em cães, sendo a intensidade da resposta amenizada durante o tratamento bem-sucedido dos pacientes; incrementos de proteína C-reativa e amilóide A sérica não estão relacionados à recidiva do linfoma; a quimioterapia do linfoma com os protocolos COP e VCM não altera a resposta de fase aguda, avaliada por meio dessas proteínas, nem existe diferença na resposta de fase aguda entre tais protocolos; existe relação direta entre os níveis de proteína C-reativa e amilóide A sérica no curso do linfoma e da quimioterapia; aumentos das concentrações séricas de proteína C-reativa são acompanhados de elevações da fração de &beta;2-globulinas e aumentos de amilóide A sérica são acompanhados de elevações de &beta;1-globulinas. / Lymphoma is a common neoplasm in dogs and chemotherapy is indicated to achieve long-term survivals. During the treatment, frequent relapses require drug regimen modifications. C-reactive protein (CRP) and serum amyloid A (SAA) are hepatic acute-phase mediators and usually are increased in inflammatory, infectious and neoplastic conditions. The aim of this study was to evaluate the role of these proteins in remission and relapse monitoring of dogs with lymphoma, under 2 chemotherapy protocols. COP protocol (cyclophosphamide, vincristine and prednisone) included an one-month induction period and maintenance cycles each 21 days and VCM protocol (vincristine, cyclophosphamide, methotrexate and L-asparaginase) was administered in a continuous weekly schedule. Five groups were composed: Normal (20 healthy dogs), COP Control (4 healthy dogs submitted to chemotherapy with COP protocol), VCM Control (4 healthy dogs submitted to chemotherapy with VCM protocol), COP Lymphoma (10 dogs with multicentric lymphoma treated with COP protocol) and VCM Lymphoma (10 dogs with multicentric lymphoma treated with VCM protocol). CRP and SAA were determined by Elisa tests and the electrophoresis was performed in cellulose acetate strips. In Normal dogs, CRP and SAA levels, as well the electrophoretic fractions, were measured only one time; in COP Control and VCM Control groups of dogs at the chemotherapy weeks 1, 2, 3, 4, 7, 10, 13 and 16; in dogs from groups COP Lymphoma and VCM Lymphoma, at the weeks 1, 2, 3 and 4, beside the relapse and a called stability moment immediately before the relapse. Results were compared by means of repeated measures variancy analyses, considering the groups and the observation weeks as the control factors, followed by Tukey´s multiple comparisons, at 5 % of significance level. It was concluded that: lymphoma induces an acute phase response in dogs and the intensity of response declines along the disease remission; increases of CRP and SAA are not related to lymphoma relapse; neither COP nor VCM chemotherapy changes the acute-phase response, when CRP and SAA are taken in account, and there is not difference on acute-phase response between both regimens; there is a positive correlation between CRP and SAA levels during lymphoma assessment and chemotherapy; increases of CRP levels are followed by &beta;2-globulin elevations and increases of SAA levels are related to &beta;1-globulin elevations.

Orientador: Priscylla Tatiana Chalfun Guimarães Okamoto / Resumo: A doença do trato urinário inferior em felinos (DTUIF) apresenta diversos fatores etiológicos, sendo a forma obstrutiva a mais grave. As proteínas de fase aguda (PFAs) são biomarcadores utilizados para avaliar processos inflamatórios sistêmicos. A Alfa-1 Glicoproteína Ácida e o Amilóide A Sérico são PFAs positivas e major, o fibrinogênio é PFA minor e a albumina é negativa em gatos. O objetivo deste estudo é determinar as concentrações séricas das proteínas de fase aguda, Amilóide A sérico, Alfa-1 Glicoproteína Ácida, fibrinogênio e albumina e utilizá-las como biomarcadores de inflamação no monitoramento do processo inflamatório de gatos com doença do trato urinário inferior obstrutiva. Foram avaliados 25 gatos, machos, sem predileção de raça e idade, divididos em dois grupos experimentais, GC - grupo controle com oito gatos hígidos e GO - grupo obstruído com 17 gatos diagnosticados com DTUIF obstrutiva. Foram coletadas amostras para determinação das PFAs, bioquímica sérica, urinálise e UP/C nos M0, M12, M24 e M48 no GO e no GC somente no primeiro momento. As determinações das PFAs foram realizadas com os kits de ELISA para SAA, Kit Cat Serum Amyloid A Elisa (LIFE-SAA-8) e AGP, Kit Cat Alpha-1-Acid Glycoprotein Elisa (LIFE-AGP-8), ambos marca: Life Diagnostics®. No M0 houve correlações positivas de SAA, AGP e fibrinogênio com ureia e creatinina e correlação negativa de albumina com hematúria, SAA e potássio. No M48, houve correlações positivas entre SAA e AGP, AGP e ureia, fi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Feline Lower Urinary Tract Disease (FLUTD) presents several etiologic factors, with the obstructive form representing the most severe. The acute phase proteins (APPs) are biomarkers used to evaluate systemic inflammatory processes. In cats, Alpha-1-Acid Glycoprotein and Serum Amyloid A are major positive APPs, fibrinogen is a minor APP and albumin is a negative APP. This study aims at determining the serum concentrations of acute phase proteins Serum Amyloid A, Alpha-1-Acid Glycoprotein, fibrinogen and albumin, in addition of using them as biomarkers of inflammation during the monitoring of the inflammatory processes of cats with obstructive feline lower urinary tract disease. A total of 25 male cats were recruited for the study irrespective of breed and age, and were divided into two experimental groups: the control group (CG), comprised of eight healthy cats; and the obstruction group (OG), comprised of 17 cats diagnosed with obstructive FLUTD. Samples were collected for APP analysis, serum biochemical assay, urinalysis and UP/C determination at M0, M12, M24 and M48 in the OG, and at M0 in the CG. The concentrations of the APPs were determined using commercially-available ELISA kits for SAA (Kit Cat Serum Amyloid A Elisa, LIFE-SAA-8) and AGP (Kit Cat Alpha-1-Acid Glycoprotein Elisa, LIFE-AGP-8) (Life Diagnostics®). At M0, there were positive correlations of SAA, AGP and fibrinogen with urea and creatinine, as well as negative correlations between albumin and hematuria, ... (Complete abstract click electronic access below) / Mestre

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

Interleukin (IL)-1beta is a powerful pro-inflammatory cytokine with important roles in directing both innate and adaptive immunity. As a result, its production is tightly controlled, with the synthesis of an inactive form (pro-IL-1beta) and the requirement of a second signal. This induces the formation of the inflammasome, a macromolecular complex which mediates the maturation of IL-1beta into the bioactive cytokine. Given its significance, it is important to identify mechanisms of IL-1beta induction and modulation. Firstly, we describe serum amyloid A (SAA), an acute phase protein with immunomodulatory properties, as a novel inducer of IL-1beta. Using cells from genetically modified mice, the molecular mechanisms responsible were dissected, demonstrating the receptors TLR2 and NLRP3 as required for this effect. By instilling SAA into mice, we also show that SAA is able to induce IL-1beta production in vivo. Invariant natural killer T (iNKT) cells have also been shown to be important modulators of immunity, mediating both pro- and anti-inflammatory responses. iNKT cells are non-conventional T lymphocytes which recognise glycolipid in the context of CD1d, with the ability to interact with immature antigen presenting cells in an autoreactive manner. We link the regulatory ability of iNKT cells with IL-1beta production, showing that a low activation signal leads to the induction of an IL-13-dominated cytokine profile, as well as weak engagement of the CD40-CD40L pathway. We show for the first time that through these mechanisms, iNKT cells are able to dampen the secretion of IL-1beta upon subsequent stimulation of dendritic cells. We hypothesise that this effect of iNKT cells is important in controlling inflammatory responses in vivo, and demonstrate exacerbated IL-1beta production and inflammation during influenza virus infection of iNKT cell-deficient animals. This novel anti-inflammatory property of iNKT cells may be harnessed in the therapeutic intervention of inflammatory disorders.

Linfomas pertencem a um grupo de neoplasias que têm em comum a origem em células linforreticulares, manifestando-se geralmente em tecidos linfóides. Em sua evolução, há uma reação generalizada do organismo de forma não específica contra as alterações sistêmicas que comprometem a homeostase, conhecida como resposta de fase aguda, a qual leva a uma importante alteração na síntese de proteínas pelo fígado, resultando no aumento de algumas proteínas conhecidas como proteínas de fase aguda, sendo as de maior relevância a amiloide sérica A, &alpha;-1 glicoproteína ácida e proteína C reativa. Foram objetivos deste estudo, definir o perfil eletroforético do felino com linfoma e avaliar as concentrações séricas de amilóide sérica A (ASA), &alpha;-1 glicoproteína ácida (GPA) e proteína C reativa (PCR) destes animais durante a quimioterapia, avaliando-se como possíveis indicadores de remissão de doença. Os grupos de estudo foram constituídos por 20 felinos clinicamente normais (controle) e 16 felinos com linfoma (experimental). Foram excluídos pacientes que apresentavam tratamentos prévios e/ou doenças concomitantes. A eletroforese das proteínas séricas foi realizada em tiras de acetato de celulose. Para as mensurações de ASA, GPA e PCR utilizaram-se kits comerciais, sendo as mesmas determinadas no grupo controle, uma única vez e, no grupo experimental, quando do diagnóstico e a cada 2 semanas durante 3 meses de tratamento. A análise estatística foi realizada com testes paramétricos, sendo o teste t não pareado utilizado para comparações entre os grupos controle e experimental no momento do diagnóstico e análise de variância simples (ANOVA), seguida do teste de comparações múltiplas de Tukey, para comparar o grupo experimental no diagnóstico e semanas de tratamento. Foram observadas diferenças significantes entre os grupos controle e experimental no momento do diagnóstico, com relação à GPA (p<0,0001), ASA (p=0,0028), PCR (p=0,0003), globulina (p=0,0087), relação albumina: globulinas (p<0,0001) e &alpha;-2 globulinas (p=0,0082). Quando se compararam os achados do grupo experimental no diagnóstico e nas semanas de tratamento houve diferença nos resultados referente à GPA (p=0,0021) e ASA (p=0,0053), enquanto os níveis de PCR não se alteraram significativamente (p=0,4510). Concluiu-se que os felinos com linfoma apresentaram uma expressiva resposta de fase aguda, caracterizada por aumento das concentrações séricas de &alpha;-1 glicoproteína ácida, amiloide sérica A e proteína C reativa, sendo a amilóide sérica A e &alpha;-1 glicoproteína ácida potenciais indicadores de remissão de doença naqueles pacientes que estavam com suas concentrações elevadas quando do diagnóstico. / Lymphoma belongs to a group of malignancies that have in common their origin in lymphoreticular cells, and is generally manifested in lymphoid tissues. In its evolution, there is a generalized reaction of the organism against a non-specific systemic conditions that compromises the homeostasis, known as acute phase response, which leads to a significant change in protein synthesis by the liver, resulting in an increase of some proteins known as acute phase proteins, and the most relevant are serum amyloid A, &alpha;-1 acid glycoprotein and C-reactive protein. This study was designed to define the electrophoretic profile of feline lymphoma and to evaluate serum concentrations of serum amyloid A (ASA), &alpha;-1 acid glycoprotein (GPA) and C-reactive protein (PCR) of these animals during chemotherapy, evaluated as possible indicators of disease remission. The study groups consisted of 20 clinically normal cats (control) and 16 cats with lymphoma (experimental). We excluded patients who had previous treatments and/or concomitant diseases. Electrophoresis of serum proteins was conducted on strips of cellulose acetate. For measurements of ASA, GPA and PCR we used commercial kits, which are then determined, in the control group, only once and, in the experimental group, at diagnosis and every 2 weeks during 3 months of treatment. Statistical analysis was performed with parametric tests, where unparied t test was used for comparisons between control and experimental groups at diagnosis and simple analysis of variance (ANOVA) test followed by Tukey\'s multiple comparisons to compare the experimental group in the diagnosis and weeks of treatment. There were significant differences between control and experimental groups at diagnosis of &alpha;-1 acid glycoprotein (p<0,0001), serum amyloid A (p=0,0028), C-reactive protein (p=0,0003), total globulin (p=0,0087), albumin: globulin (p<0,0001) and &alpha;-2 globulins (p=0,0082). When comparing the experimental group in the diagnosis and weeks of treatment was significant in the results of &alpha;-1 acid glycoprotein (p=0,0021) and serum amyloid A (p=0,0053), whereas C-reactive protein did not change significantly (p=0,4510). It is concluded that cats with lymphoma have an expressive acute phase response, characterized by increased in serum concentrations of serum amyloid A, &alpha;-1 acid glycoprotein and C-reactive protein, and the serum amyloid A and alpha-1 acid glycoprotein reference potential indicators of remission in those patients who were with their high concentrations at diagnosis.

INTRODUÇÃO: Sepse e choque séptico são doenças comuns em pacientes gravemente enfermos, evoluindo muitas vezes com síndrome de disfunção de múltiplos órgãos (SDMO) e morte. Este trabalho investiga a eficácia da administração precoce de dexametasona em evitar a progressão do choque séptico para SDMO e morte e o comportamento da proteína amilóide A sérica (SAA) e da proteína C-reativa (PCR) como marcadores da evolução e gravidade dos pacientes em choque séptico no período pós-operatório. MÉTODOS: Estudo prospectivo, aleatório, duplamente encoberto, realizado na Unidade de Terapia Intensiva pós-operatória do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, com 29 pacientes que no período pós-operatório evoluíram com choque séptico. Os participantes foram divididos de forma aleatória em dois grupos, de acordo com a solução administrada: dexametasona 0,2 mg/kg (grupo D) ou placebo (grupo P), repetidas a cada 36 horas. Os pacientes foram acompanhados durante sete dias de internação na UTI através do escore SOFA (Sequential Organ Failure Assessment) e dosagens séricas diárias de PCR, SAA e lactato. RESULTADOS: Os pacientes do grupo D, quando comparados aos pacientes do grupo P, permaneceram mais dias sem necessidade do uso do vasopressor (respectivamente 2,9±2,7 e 0,7±0,6, p=0,01) e mais tempo livre de ventilação mecânica (respectivamente 2,6±2,5 e 0,6±0,5, p=0,03). A mortalidade no grupo P foi de 67% (10 em 15) e no grupo D foi de 21% (3 em 14) (Risco Relativo=0,31, IC95% 0,11-0,88). Os valores de PCR e SAA apresentaram forte correlação durante o período de observação (R=0,91/p=0,002). PCR e SAA não tiveram correlação com o escore SOFA (respectivamente R=0,71/p=0,05 e R=0,52/p=0,18), nem com o lactato (p=0,88 e p=0,67). CONCLUSÕES: O tratamento precoce com dexametasona nos pacientes com choque séptico reduziu a mortalidade em 7 dias de acompanhamento. Os níveis séricos de PCR e SAA apresentaram-se elevados nos pacientes em choque séptico e tiveram forte correlação, porém não foram preditores de disfunção orgânica nem de mortalidade. / INTRODUCTION: Sepsis and septic shock are a very common condition in critically ill patients, leading to multiple organ dysfunction syndrome (MODS) and death. This study aimed at investigating the efficacy of early administration of dexamethasone in patients with septic shock in order to block the evolution to MODS and death. It also evaluated serum amyloid A protein (SAA) and C-reactive protein (CRP) as evolution and organ dysfunction markers in postoperative septic shock patients. METHODS: Prospective, randomized, double-blind study, developed in a surgical intensive care unit of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo that involved 29 patients with septic shock. All eligible patients were prospectively randomized to receive either a dose of 0.2 mg/kg of dexamethasone (group D) or placebo (group P), repeated every 36 hours intervals. Patients were monitored over a 7- day period by Sequential Organ Failure Assessment score (SOFA) and daily measurements of CRP, SAA and lactate. RESULTS: Patients treated with dexamethasone had more vasopressor therapy-free days (2.9±2.7 versus 0.7±0.6, p=0.01) and more mechanical ventilation-free days (2.6±2.5 e 0.6±0.5, p=0.03). Mortality in group P was 67% (10 in 15) and in group D was 21% (3 in 14) (Relative Risk=0.31, 95%CI 0.11 to 0.88). CRP and SAA were strongly correlated during the 7 day period of observation (R=0.91/p=0.002). CRP and SAA did not correlate with SOFA (respectively R=0.71/p=0.05 and R=0.52/p=0.18) and lactate (p=0.88 and p=0.67). CONCLUSIONS: Early treatment with dexamethasone reduced 7-day mortality in septic shock patients. CRP and SAA levels were significantly elevated in septic shock and were strongly correlated to each other, but did neither correlate with organ dysfunction nor predict mortality.

Nos últimos anos, nosso grupo de pesquisa vem descrevendo vários efeitos da SAA em células do sistema imune no que diz respeito à expressão e liberação de citocinas pró-inflamatórias. Neste estudo centramos nossa atenção na verificação dos efeitos da SAA sobre células mononucleares. Para isto, usamos três modelos experimentais. Em murinos, descrevemos a habilidade da SAA induzir a produção de NO por macrófagos peritoneais e, com uso de animais knockout para TLR4, sugerimos que SAA seja um ligante endógeno do TLR4. Em células mononucleares de sangue periférico humano, a SAA induz a expressão e liberação de CCL20, uma quimiocina importante na transição da resposta imune inata para adaptativa, bem como a expressão dos fatores M-CSF e VEGF. Em células THP-1, mostramos a cinética de fosforilação de proteínas tirosina quinases promovida pela SAA e comparamos com LPS, um estímulo pró-inflamatório clássico. Ainda em células THP-1 mostramos que a SAA induz a fosforilação de duas proteínas importantes no processo inflamatório por induzirem a ativação de NF&#954;B; a p38 e a ERK1/2. Com este estudo contribuímos com o conhecimento a respeito do papel regulatório da SAA em células mononucleares. A ação da SAA sobre estas células torna-se importante, pois estas são cruciais na resposta imune inata e também atuam como células acessórias na resposta imune adaptativa. Desta forma, evidencia-se que, no processo de fase aguda, a expressão e a síntese de SAA resultam na modulação de etapas que controlam este processo e sua progressão. / In the past few years, our research group has described various effects of serum amyloid A (SAA) on cells of the immune system regarding the expression and release of pro-inflammatory cytokines. In this study we have focused on the effects of SAA on mononuclear cells. In order to do this, we have used three experimental models. In the murine experimental model, we described SAA\'s ability to induce the production of NO through peritoneal macrophages and, by using knockout animals for TLR4, we suggested that SAA is an endogenous agonist of TLR4. In mononuclear cells of peripheral human blood, SAA induced the expression and release of CCL20, an important chemokine in the transition from the innate to the adaptive immune response, as well as the expression of M-CSF and VEGF-factors. In THP-1 cells, we showed the phosporylation kinetics of tyrosine protein kinases induced by SAA, and we compared it to LPS, a classic pro-inflammatory stimulus. We also demonstrated, in THP-1 cells, that SAA induced the phosphorylation of two proteins, namely p38 and ERK1/2, that are crucial in the inflammatory process because they induce the activation of transcription factors. With this study, we contributed to the knowledge of the regulatory role of SAA in mononuclear cells. Activity of SAA on these cells is highly important, for they are crucial in the innate immune response and act as accessory cells in the adaptive immune response. Hence it is evident that, in the acute phase process, the expression and synthesis of SAA result in the modulation of the phases that control this process and its progression.

Amyloidoses comprise a group of gain-of-toxic function protein misfolding diseases, in which normally soluble proteins in their functional state undergo conformational changes into highly organized and generally intractable thread-like aggregates, termed amyloid fibrils. These structures accumulate predominantly in the extracellular space but growing evidence suggests that amyloids may start to form intracellularly. At least 26 different human proteins, intact or in fragmented form, are known to form amyloid, which is linked with many debilitating neurodegenerative diseases such as Alzheimer’s disease (AD), Creutzfeldt-Jakob disease, and transthyretin (TTR)-related amyloidosis (ATTR). In this work, we focus on ATTR, which is one of the most frequent systemic amyloid diseases. A functional link was established between hereditary ATTR, a severe and fatal disorder and the enhanced propensity of the human plasma protein transthyretin (TTR) to form aggregates, caused by single point mutations in the TTR gene. The disease is heterogeneous and clinical symptoms vary from cardiomyopathy to progressing sensorimotor polyneuropathy depending on TTR variant involved and the amyloid deposition site. Despite the fact that TTR-derived amyloid accumulates in different organs such as heart, kidney, eyes, and predominantly in the peripheral nerves of ATTR patients, the exact mechanism of the disease development is not understood. In contrast to the case of AD, it has been difficult to generate an animal model for ATTR in transgenic mice that would be useful in understanding TTR aggregation processes and the mechanisms of the associated toxicity as these mice did not develop any neuropathic phenotype besides amyloid deposits. Therefore, we created a disease-model in Drosophila due to its huge repertoire of genetic techniques and easy genotype – phenotype translation, as well as its success in modeling human neurodegeneration. We have generated transgenic flies that over-express the clinical amyloidogenic variant TTRL55P, the engineered variant TTR-A (TTRV14N ⁄ V16E), and the wild-type protein. All TTR variants were found in the secreted form in the hemolymph where misfolding occurred and depending on the pool of toxic species, the fate of the fly was decided. Within a few weeks, both mutants (but not the wild-type TTR) demonstrated a time-dependent aggregation of misfolded molecules in vivo. This was associated with neurodegeneration, change in wing posture, attenuation of locomotor activity including compromised flying ability, and shortened life span. In contrast, expression of the wild-type TTR had no discernible effect on either longevity or fly behavior. In this work, we also addressed the correlation between TTR transgene dosage and thus, protein levels, with the severity of the phenotypes observed in TTR-A flies which developed a “dragged wing” phenotype. Remarkably, we established that degenerative changes such as damage to the retina strictly correlated with increased levels of mutated TTR but inversely with behavioral alterations and the dragged wing phenotype. We characterized formation of aggregates in the form of 20 nm spherules and amyloid filaments intracellularly in the thoracic adipose tissue and brain glia (both tissues that do not express the transgene). Moreover, we detected a fraction of neurotoxic TTR-A in the hemolymph of young but not old flies. We proposed that these animals counteract formation and persistence of toxic TTR-A species by removal from the circulation into intracellular compartments of glial and fat body cells and this is part of a mechanism that neutralizes the toxic effects of TTR. We validated the fly model for ATTR by applying a genetic screen during study of modifier genes. We found Serum amyloid P component (a product of the APCS gene) as a potent modifier of TTR amyloid-induced toxicity that was effective in preventing the apoptotic response in cell culture assay and capable of reducing the dragged wings when co-expressed in TTR-A flies. Finally, we optimized this fly model in order to screen for therapeutic compounds effective against ATTR. Feeding assays showed the effectiveness of several compounds among known native-state kinetic stabilizers of TTR against its aggregation. We described several early endpoints in this model, which can be used as a rapid and cost-effective method for optimizing concentrations and pre-screening of drug candidates. As the proof of principle, by feeding flies with increasing doses of diflunisal analogue (an FDA-approved Non-Steroidal Anti-Inflammatory Drug) a dose-dependent reduction of the dragged wings was observed.