Orientador: Maria Heloisa Souza Lima Blotta / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T06:51:40Z (GMT). No. of bitstreams: 1 Corvino_ClaudiaTres_M.pdf: 633875 bytes, checksum: 46aff21f882b64dcbb4eefa73f93a232 (MD5) Previous issue date: 2005 / Resumo: A interleucina 18 (IL-18) é uma citocina pró-inflamatória com importante participação tanto na resposta inata como adquirida. Concentrações elevadas de IL-18 são encontradas em várias doenças inflamatórias e infecciosas. A expressão aumentada de IL-18 pode contribuir para a resolução de processos infecciosos, mas também pode promover uma resposta inflamatória exagerada, prejudicial ao hospedeiro. O objetivo deste estudo foi determinar as concentrações séricas de IL-18 e outros mediadores inflamatórios (IL-12, sICAM-1, sTNFRI, sTNFRII, CXCL9, CXCL10), antes e após tratamento antifúngico, em pacientes com a forma juvenil (FJ) e adulta (FA) da paracoccidioidomicose (PCM), assim como em indivíduos normais saudáveis e relacionar com a atividade e gravidade da doença. Também foram avaliadas as concentrações de IgG total, subclasses de IgG e IgE anti-gp43 de P. brasiliensis. Todas as dosagens foram realizadas por método imunoenzimático (ELISA). Os níveis séricos basais de IL-18, IL-12, sICAM-1, sTNFRI, sTNFRII, CXCL9, CXCL10 estavam mais elevados em pacientes com PCM em relação ao grupo controle. Pacientes com a FJ apresentaram concentrações mais elevadas de IL-18 e sTNFRII em relação aqueles com a FA da PCM, enquanto estes mostraram maiores concentrações de sTNFRI em relação aos pacientes com a FJ. Houve correlação positiva entre as concentrações de IL-18 e sICAM-1 (r=0.62, p<0.0001), sTNFRI (r=0.61, p<0.0001), sTNFRII (r=0.64, p=0.002) e também em relação a gravidade da doença nos pacientes com a FJ. A terapia anti-fúngica resultou em significante diminuição das concentrações séricas de todos os mediadores inflamatórios analisados que, de maneira geral, atingiram os valores encontrados no grupo controle ao final de 2 anos de tratamento. A pesquisa de anticorpos mostrou níveis basais maiores de IgG4 e IgE nos pacientes com a FJ, enquanto que a IgG1 estava mais elevada naqueles com a FA. Em conjunto os resultados mostraram que uma forte resposta inflamatória marca a fase inicial da PCM humana e que mediadores como a IL-18 e o sTNFRII parecem contribuir em particular para uma forma mais grave da doença / Abstract: IL-18 is a pro-inflammatory cytokine of the IL-1 superfamily that exhibits broad functional effects in innate and acquired immune responses and which has been found in high levels in several chronic inflammatory and autoimmune diseases. Over-expression of IL-18 may promote early resolution of infection or could induce a detrimental exaggerated immune response. The aim of this study was to determine serum levels of IL-18 and other inflammatory mediators (IL-12, sICAM-1, sTNFRI, sTNFRII, CXCL9, CXCL10) at baseline and after antifungal therapy in serum from patients with juvenil (JF) and adult form (AF) of paracoccidioidomycosis (PCM) as well as in healthy controls (C), and to assess their possible relationships to the severity of disease. IL-18 and sTNFRII levels in patients with the JF of PCM were significantly higher than in the AF and controls. In relation to sICAM-1, no difference was observed between JF and AF but both presented higher levels than controls. sTNFRI levels were higher in patients with PCM than in controls, and significant higher concentrations were detected in AF patients as compared to JF ones. Moreover IL-12 and chemokines CXCL9 and CXCL10 were also higher in patients than in controls. In JF patients IL-18 levels significantly correlated with sICAM-1 (r=0.62, p<0.0001), sTNFRI (r=0.61, p<0.0001), sTNFRII (r=0.64, p=0.002), as well as with clinical severity. The results suggest the value of serum IL-18 and sTNFRs levels as a parameter of PCM severity and may support a possible role for them in the pathogenesis of the disease / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A Esquistossomose à uma doenÃa causada por parasitos do gÃnero Schistosoma matando centenas de milhares de pessoas por ano no mundo. O Schistosoma tem vÃrias espÃcies com interesse clÃnico. No Brasil o causador da Esquistossomose à o S. mansoni e o principal hospedeiro e reservatÃrio do parasito à o homem sendo a partir desse que os ovos sÃo eliminados nas fezes. Os hospedeiros intermediÃrios sÃo os caramujos. As principais espÃcies de caramujos hospedeiros do Schistosoma mansoni no Brasil sÃo: Biomphalaria glabrata, Biomphalaria tenagophila e Biomphalaria straminea. Sendo a terceira espÃcie a predominante no CearÃ. A doenÃa tem presenÃa constante em mais de 74 paÃses (praticamente todos subdesenvolvidos). Cerca de 500 a 600 milhÃes de pessoas correm riscos de serem atingidas e mais de 200 milhÃes sÃo infectadas a cada ano, e isso se deve principalmente a falta de saneamento bÃsico e educaÃÃo sanitÃria. Para a melhor profilaxia desta doenÃa, deve ser feito o diagnÃstico e o tratamento da populaÃÃo, orientando para evitar entrar em contato com Ãguas que contenham caramujos (aÃudes, lagos, lagoas, rios etc). à uma doenÃa que pode evoluir para complicaÃÃes graves, eventualmente levando ao Ãbito em funÃÃo de extensa fibrose decorrente da presenÃa em parÃnquima hepÃtico de ovos do Schistosoma mansoni, formando granulomas. O principal objetivo desse estudo foi desenvolver uma estratÃgia para aumentar a eficÃcia na identificaÃÃo de pacientes infectados com S.mansoni, em Ãrea de baixa endemicidade, no Estado do CearÃ, usando um protocolo combinando uma tÃcnica sorolÃgica (IgG â ELISA) com exames de fezes seqÃenciais. Esse trabalho foi realizado em etapas, no qual na primeira, dos 287 indivÃduos que realizaram o mÃtodo de Kato-Katz, 11 (3,8%) apresentaram resultados positivos para S. mansoni. Com relaÃÃo aos outros helmintos foram encontrados: Trichuris trichiura 25 (8,7%), Ascaris lumbricoides 19 (6,6%) e Ancilostomideos 15(5,2%). Em relaÃÃo ao testes de ELISA, 97 (33,8%) foram positivos. Todos os pacientes que apresentaram ovos nas fezes foram positivos no teste sorolÃgico. Neste grupo estÃo inclusos os 11 que foram positivos na coproscÃpia. Dos pacientes com ELISA positivo e Kato-Katz negativos, apenas 56 entregaram as trÃs amostras de fezes para uma segunda anÃlise coproscÃpica. Desses, 14 (25%) foram positivos para Schistosoma mansoni. Dos 42 pacientes que continuavam negativos, 22 responderam no questionÃrio que nunca tiveram esquistossomose como tambÃm nunca receberam tratamento para a doenÃa. O presente estudo nÃo trata sà de determinar a prevalÃncia da doenÃa no municÃpio, e sim de identificar o maior nÃmero possÃvel de indivÃduos infectados, usando o mÃtodo sorolÃgico que foi aplicado de forma a contemplar a populaÃÃo residente em Ãreas de risco de transmissÃo ou expostas ao risco de infecÃÃo, principalmente por atividades domÃsticas e de lazer. Diante destes resultados, acredita-se, em concordÃncia com outros autores, que utilizando a tÃcnica de ELISA combinado com anÃlises repetidas de no mÃnimo 5 lÃminas de fezes, torna-se mais fÃcil diagnosticar pacientes com a esquistossomose, melhorando assim a hipÃtese que provavelmente em um futuro prÃximo, seremos capazes de combinar mÃtodos parasitolÃgicos e sorolÃgicos no programa de controle da esquistossomose, um fator importante para detecÃÃo de novos portadores da doenÃa. / Schistosomiasis is a disease caused by parasites of the genus Schistosoma, killing hundreds of thousands of people each year worldwide.. The Schistosoma has several species of clinical interest. In our country the cause of Schistosomiasis is the S. mansoni and the main reservoir host and the parasite is starting with the man that the eggs are removed in feces. The snails are the intermediate host. The main species of snails host of Schistosoma mansoni in Brazil are: Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea. The third kind in the predominant in CearÃ. The disease has presence in over 74 countries (virtually all underdeveloped). Around state 500 to 600 million people are at risk of being affected and more than 200 million are infected every year, and this is mainly due to lack of sanitation and health education. To the best prevention of this disease is to be made the diagnosis and treatment of the population to avoid targeting comes in contact with water containing snails (ponds, lakes, rivers etc). It is a disease that can develop into serious complications, possibly leading to death according to extensive fibrosis caused by the presence in liver parenchyma of the Schistosoma mansoni eggs, forming granulomas. So the main objective of this study was to develop a strategy to increase effectiveness in identifying patients positive for Schistosomiasis in areas of low endemic in the state of Ceara, using a protocol combining a technique in which antibodies (IgG - ELISA) with examinations of sequential stool. This work was followed by steps, in which the first of the 287 patients who underwent the method of Kato-Katz, 11 (3.8%) showed positive results for S. mansoni. On the other helminths are: Trichuris trichiura 25 (8.7%), A. lumbricoides 19 (6.6%) and Hookworms 15 (5.2%). For the tests, ELISA, 97 (33.8%) were positive. All patients who had eggs in the feces were positive in the serologic test. In this group are included the 11 that were positive in feces analysis (Figure 1). From patients with Elisa positive and negative Kato-Katz, only 56 handed the three samples of stool for a second analysis Of these, 14 (25%) were positive for Schistosoma mansoni. Of the 42 patients who remained negative, 22 responded in the questionnaire that had never schistosomiasis but never received treatment for the disease. Our present study was to not only determine the prevalence of the disease in the municipality, and to identify the largest possible number of infected individuals, the serological method was applied in order to accommodate the population living in those areas of risk of transmission or at risk of infection, mainly by domestic and leisure activities. In view of our results, we believe, in agreement with other authors, that using the ELISA technique combined with repeated analysis of at least 5 simples of feces, it becomes easier to diagnose patients positive for schistosomiasis, thus improving the hypothesis that probably in the near future, being able to combine parasitological and sorological in the programme for the control of schistosomiasis, an important factor for detection of new carriers of the disease.

Os objetivos desse estudo foram: 1) Padronizar a pesquisa do anti-tTg, comparando-o com o anticorpo antiendomísio (AAE) e 2) Avaliar as interações da tTg com a fibronectina. 49 celíacos e 124 controles com AAE negativo foram avaliados. O AAE foi pesquisado por imunofluorescência indireta e a reatividade contra a tTg e a fibronectina por ELISA in house e com kits comerciais. O antitTg foi positivo em 46,9% e 100% dos celíacos com o ELISA in house e com kits comerciais, respectivamente. A adição de fibronectina não melhorou a sensibilidade do ELISA. Em conclusão: a detecção do antitTg por ELISA apresenta percentual elevado de falso-positivos, não podendo substituir a pesquisa do AAE / The aims of the current study were: to standardize the detection of anti-tTg antibodies, comparing them with antiendomysial antibodies (EMA) and to assess the interaction of tTg with fibronectin. 49 celiac patients and 124 controls were enrolled. EMA was detected by indirect immunofluorescence reaction and tTg and fibronectin reactivity by in house ELISA and with commercially available kits. Seropositivity to anti-tTG was found in 46.9% and 100% of patients by the in house technique and by commercial kits, respectively. Fibronectin addition did not improve the ELISA sensibility. In conclusion, ELISA for anti-tTG detection has a high rate of false positive results and does not replace EMA

Orientador: Marcia Carneiro Valera / Banca: Cláudio Antonio Talge Carvalho / Banca: Flaviana Bombarda de Andrade / Resumo: Esta pesquisa clínica teve como objetivo comparar a carga e composição microbiana bem como as concentrações de LPS e LTA encontradas na infecção endodôntica primária (IEP) e na infecção endodôntica secundária (IES). Além disso, a correlação desses achados com características clínicas e tomográficas também foram investigadas. Sessenta dentes de pacientes com IEP (31) e IES (29) foram submetidos à avaliação clínica e tomográfica, seguido do tratamento endodôntico ou retratamento. Amostras foram coletadas de cada canal radicular utilizando cones de papel. Logo após a abertura coronária (IEP) ou após a desobturação dos canais (IES) o conteúdo coletado foi submetido à técnica de cultura microbiológica para determinar a carga microbiana de bactérias anaeróbias e ao método Checkerboard DNA-DNA hybridization para investigação de espécies bacterianas presentes. O teste de Lisado de Amebócito de Limulus e o Ensaio de Imunoabsorção Enzimática foram utilizados para quantificar os níveis de LPS e LTA. Os dados obtidos foram correlacionados com os achados clínicos e tomográficos. Maiores quantidades de bactérias cultiváveis e de LPS foram encontradas na IEP (p < 0,05). Não houve diferença nos níveis de LTA entre IEP e IES (p > 0,05). A mediana de espécies por canal radicular encontrada na IEP foi de 9 espécies e na IES foi de 22 (p < 0,05). As espécies bacterianas mais prevalentes detectadas na IEP foram P. gingivalis (14/31) e S. intermedius (14/31). Na IES, as espécies mais prevalentes f... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This clinical research aimed to compare the microbial load and composition as well as the LPS and LTA concentrations found in Primary Endodontic Infection (PEI) and Secondary Endodontic Infection (SEI). In addition, the correlation of these findings with clinical and tomographic features was also investigated. Sixty patients' teeth with PEI (31) and SEI (29) were submitted to clinical and tomographic assessment, followed by endodontic treatment or retreatment. Samples were taken from each root canal using paper points. After the coronary opening (PEI) or after the removal of root filling material (SEI), the collected samples were submitted to the microbiological culture technique to determine the microbial load of anaerobic bacteria and to the Checkerboard DNA-DNA hybridization method for investigation of present bacterial species. The Limulus amebocyte lysate assay and enzyme-linked immunosorbent assay were used to quantify LPS and LTA levels. The data obtained were correlated with clinical and tomographic findings. A higher number of cultivable bacteria and LPS was found in PEI (p < 0.05). There was no difference in LTA levels between PEI and SEI (p> 0.05).The median number of species per root canal found in PEI was 9 and 22 in SEI (p < 0.05). The most prevalent bacterial species detected in PEI were P. gingivalis (14/31) and S. intermedius (14/31). In SEI, the most prevalent species were P. gingivalis (21/29) and C. rectus (20/29). LPS was positively correlated with a larg... (Complete abstract click electronic access below) / Mestre

To determine the origin of non-vagal afferent fibers innervating the heart of guinea pigs, capsaicin was injected into the ventricular myocardium to induce depletion of substance P (SP). The lower cervical, upper thoracic and lumbar spinal ganglia, as well as the left atrium and base of ventricles, were assayed for SP depletion by using the enzyme-linked immunosorbent assay (ELISA) and immunohistochemical procedures. Capsaicin affected spinal ganglia from the 3 regions differently. The substance P level in lumbar spinal ganglia remained fairly constant, while the level of SP from cervical and thoracic regions declined significantly. At the maximal depletion dosage (173 μg of capsaicin/kg), SP concentration decreased 72.3% in cervical spinal ganglia, 45.5% in thoracic ganglia and 56.1% in the atrium. The lack of SP depletion in lumbar ganglia indicates that capsaicin acted locally on cardiac afferents rather than systemically. Data from this study suggest that capsaicin-sensitive neurons of the heart have cell bodies in the lower cervical spinal ganglia as well as in the upper thoracic spinal ganglia.

Lipid transfer inhibitor protein (LTIP) is an important regulator of cholesteryl ester transfer protein function. We report the development of an immunoassay for LTIP and its use to quantify LTIP in plasma of varying lipid contents. A rabbit antibody against bacterially produced recombinant LTIP detected two LTIP isoforms in plasma differing in carbohydrate content. This antibody was used in a competitive, enzyme-linked immunoassay that uses partially purified LTIP bound to microtiter plates. To optimize LTIP immunoreactivity, plasma samples required preincubation in 1% Tween-20 and 0.5% Nonidet P-40. In normolipidemic plasma, LTIP averaged 83.5 mg/ml. LTIP was 31% higher in males than in females. LTIP was positively associated with HDL cholesterol in normolipidemic males but not in females. In hypertriglyceridemic males, LTIP was only 56% of control values, whereas in hypertriglyceridemic females, LTIP tended to increase. Additionally, in males with normal cholesterol and triglyceride (TG) ≤ 200 mg/dl, LTIP varied inversely with plasma TG. Overall, we have confirmed the negative association between plasma TG levels and LTIP previously suggested by a small data set, but now we demonstrate that this effect is seen only in males. The mechanisms underlying this gender-specific response to TG, and why LTIP and HDL levels correlate in males but not in females, remain to be determined.

A new enzyme activity assay has been developed for the target N8-acetylspermidine deacetylase, a not-well-studied but essential enzyme in the polyamine interconversion and reutilization pathway. The enzyme assay, based on mass spectrometric detection of a specific reaction product following sample introduction by flow injection, was shown to have a sensitivity of smaller than 1 micromolar and typical RSD of 3-10%. The linear range for analyte was from 1 μM to 100 μM, with R2 > 0.992. The new assay avoids the use of radio labels. Sample preparation is straightforward, and high specificity is provided by the selected reaction monitoring, SRM, using a triple quadrupole mass spectrometer. Acetylputrescine was used for the first time as the substrate for the assay of N8-acetylspermidine deacetylase in lieu of N8 -acetylspermidine. The crude enzyme extracted from rat liver had an apparent Km value of 80.6 μM for acetylputrescine and a Vmax of 1.1 nmol mg-1 min-1. Enzyme extracted from frozen rat liver was compared with that from fresh rat liver. Frozen rat liver extraction had similar kinetics parameters with the fresh preparation and had a specific activity of 0.8 nmol mg-1 min-1. N8-acetylspermidine deacetylase was partially purified by protein precipitation and gell filtration chromatography. Affinity chromatography was tentatively applied for further isolation of the enzyme, but was not yet successful.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19770号 / 農博第2166号 / 新制||農||1040(附属図書館) / 学位論文||H28||N4986(農学部図書室) / 32806 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 保川 清, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

This thesis describes the development of a way to study the N8 - acetylspermidine deacetylase enzyme activity. The method created in this thesis emphasizes sensitivity, accuracy and safety. In this study, HeLa cells were cultured and extracted to yield a crude N8 - acetylspermidine deacetylase enzyme mixture. By measuring the decrease of N8 - acetylspermidine and the increase of spetmidine, N8 -acetylspermidine deacetylase enzyme activity can be determined using either a Varian 1200L LC/MS/MS or an API 3000 LC-ES (+)/MS/MS. An acetylation-derivatization method was developed because N8 -acetylspermidine and spermidine are hard to purify from a biological sample since they are not retained on a CIS solid phase extraction column or on a RP HPLC (high performance liquid chromatography) reverse phase column due to their small molecular weight and high polarity. The quantitation of N8 -acetylspem1indine over the range 2ng/ul to 5pg/ul was fit by linear regression as y = 1.064x + 0.218 with an R-squared value of 0.9996, where y is the peak area of the fragment-ion SRM (selected reaction monitoring: m/z: 188/114) chromatograms from N8 -acetylspermindine and x is the concentration of N8 - acetylspermindine. Acetylation of spermidine (SPD) and N8-acetylspermidine (N8AcSPD) with d6-acetic anhydride produces the d9 labeled triacetylated derivative of SPD and d6 labled triacetylated spermidine derivative of N8AcSPD. These triacetylated forms are retained on a C18 column. MS/MS gives characteristic m/z fragment ions for the derivatized species: N8AcSPD (278 to 215), NlAcSPD (278 to 218) and SPD (281 to 218). The fragment-ion SRM (selected reaction monitoring) chromatograms are used for the quantitation. A plot of peak area ratios for known mixtures of N8AcSPD and total SPD versus the molar ratios of N8AcSPD and total SPD was found to fit a linear regression line withy= 0.705x + 0.035 with an R-squared value of 0.9919. Quantitation of d6- and dg-tri-acetylspermidine by LC/MS/MS is possible at the low levels of materials found in cell extracts since the separation method results in a lower limit of quantitation. This approach enables the study of N8 - acetylspermidine deacetylase enzyme activity.

Platinum-Group Metal (PGM) nanostructures as advantageous alternatives to natural peroxidases have drawn great attention because of their superior catalytic activities, which can effectively enhance performance of enzyme-based in vitro diagnostics. The catalytic activity of metal nanoparticle peroxidase mimics can depend on their size, shape, elemental composition, and surface ligand of PGM nanostructures. Therefore, to develop optimal peroxidase mimics for a few bioanalytical and diagnostic applications, such as enzyme-linked immunosorbent assay (ELISA), it is important to investigate how structural aspects of PGM nanoparticles correlate with the ability of the nanoparticles to serve as functional mimics of protein peroxidase enzymes. In summary, this dissertation has studied: 1) iridium (Ir), platinum (Pt) and Ir/Pt bimetallic nanowire structures as peroxidase mimics, and the effect of different wires' length on their peroxidase-like activities and certain application of sandwich ELISA for the detection of carcinoembryonic antigen (CEA, a cancer biomarker); 2) ultra-small Ir nanoparticles, with an average size of 1.1 nm, supported by WO2.72 nanowire with high catalytic activity. Those Ir nanoparticles were applied to sandwich ELISA and competitive ELISA for sensitive detection of CEA and aflatoxin B1 (AFB1, a carcinogenic toxin), respectively; 3) the size effect of peroxidase mimics on their catalytic activities and performance in biosensing application, where Pd-Ir core-shell nanoparticles were used as a type of model peroxidase mimics. These studies may significantly stimulate further investigations of PGM nanostructures as peroxidase mimics and other potential applications in in vitro diagnostics.