Orientador: Luciane Dias de Oliveira / Banca: Antonio Olavo Cardoso Jorge / Banca: Mariella Vieira Pereira Leão / Resumo: A proposta deste trabalho foi avaliar a efetividade de extratos naturais, como soluções irrigadoras, sobre E. faecalis, S. aureus, E. coli, e endotoxinas (LPS) em canais radiculares. Foi avaliado também o efeito citotóxico do conteúdo do canal, após os tratamentos, pela produção de TNF-α e IL-1β em cultura de macrófagos. Foram utilizados 84 dentes unirradiculados, contaminados com associação dos três microrganismos por 28 dias. Após, foram divididos em 7 grupos (n=12), de acordo com o irrigante utilizado: G1) solução fisiológica (controle); G2) solução de clorexidina 2%; G3) extrato glicólico de própolis 12%; G4) extrato glicólico de alcaçuz 20%; G5) extrato glicólico de barbatimão 20%; G6) extrato glicólico de romã 20%; G7) extrato glicólico de cavalinha 20%. Foram realizadas 2 coletas do conteúdo do canal radicular: imediatamente após a instrumentação e após 7 dias da instrumentação. Para todas as coletas, foi realizada avaliação da atividade antimicrobiana pela contagem de UFC/mL em meios de cultura seletivos, verificação da neutralização de endotoxinas pelo teste cinético cromogênico do lisado de amebócitos de Limulus (LAL) e avaliação dos efeitos citotóxicos do conteúdo do canal pela produção de IL-1 e TNF-α por macrófagos. Os resultados foram analisados estatisticamente (ANOVA e teste de Tukey, 5%). Todos os extratos promoveram significativa redução microbiana em relação ao controle na 1ª coleta após instrumentação (p<0,05). Com relação E. faecalis, os extratos de própolis (G3), barbatimão (G5), romã (G6) e cavalinha (G7) promoveram ausência de crescimento microbiano em muitos espécimes, sendo semelhantes a clorexidina e diferentes dos demais grupos (p<0,05). Com relação S. aureus, os extratos de própolis (G3) e barbatimão (G5) promoveram ausência de crescimento microbiano em todos os espécimes, sendo semelhantes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this study was to evaluate the effects of natural extracts as irrigating solutions on E. faecalis, S. aureus, E. coli, and endotoxins (LPS) in root canals. The post treatment citotoxic effect of root canal content was also evaluated by TNF-α and IL-1β production by macrophages cell culture. A total of 84 single root canal teeth were contaminated with E. faecalis, S. aureus and E. coli for 28 days. The groups were assigned into 7 groups (n=12) according to the irrigant solution used: G1) physiologic solution (control); G2) chlorhexidine 2%; G3) propolis glycolic extract 12%; G4) liquorice glycolic extract 20%; G5) barbatiman glycolic extract 20%; G6) pomegranate glycolic extract 20%; G7) horsetail glycolic extract 20%. Samples were twice collected from root canals: immediately after instrumentation and 7 days after instrumentation. All samples were submitted to antimicrobial evaluation by CFU/mL counting on selective culture media. The endotoxin detoxification was evaluated by Limulus amebocyte lysate assay (LAL), and the citotoxic effects was evaluated by TNF-α and IL-1β production by macrophages cell culture. The results were statistically analysed (ANOVA and Tukey, 5%). All the extracts promoted significant microbial reduction compared to the control group on samples collected immediately after instrumentation (p<0,05). E. faecalis results showed that propolis (G3), barbatiman (G5), pomegranate (G6) and horsetail (G7) extracts promoted no microbial growth in all samples, similar to chlorhexidine solution and different from other groups (p<0,05). S. aureus results showed that propolis (G3) and barbatiman (G5) promoted absence of microbial growth in all samples, similar to chlorhexidine solution and different from other groups (p<0,05). E. coli results showed that propolis (G3), pomegranate (G6) and horsetail (G7) promoted... (Complete abstract click electronic access below) / Mestre

Orientador: Brenda Paula Figueiredo de Almeida Gomes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-09T19:55:04Z (GMT). No. of bitstreams: 1 Vianna_MorganaEli_D.pdf: 1593735 bytes, checksum: f9177ad362c4724b87ff726decdb4f8f (MD5) Previous issue date: 2006 / Resumo: O objetivo deste estudo foi avaliar a redução microbiana e de endotoxinas após o preparo químico-mecânico (PQM) e uso de medicamentos intracanais (MI) e a susceptibilidade de algumas espécies bacterianas frente a antimicrobianos. As amostras foram obtidas de dentes com polpa necrosada e lesão periapical, em diversas etapas do tratamento. Foram utilizados meios de transporte, cultura e incubação que proporcionaram o crescimento de microrganismos anaeróbios estritos. Os microrganismos foram identificados por testes bioquímicos padronizados e as endotoxinas quantificadas pelo teste Lisado de Amebócito de Limulus (LAL). O E-test foi utilizado para avaliar a susceptibilidade dos agentes antimicrobianos: benzilpenicilina, amoxicilina, eritromicina, azitromicina, cefaclor, amoxicilina+ácido clavulânico, metronidazol e clindamicina. Os resultados mostraram que as amostras coletadas dos canais radiculares com lesão periapical apresentaram microrganismos em todos os casos, as unidades formadoras de colônia (UFC/mL) variaram de 4x105 a 2,6x106. Os microrganismos mais encontrados foram: Propionibacterium acnes, Gemella morbillorum, Actinomyces naeslundii e Eubacterium lentum. As endotoxinas estavam presentes em todas amostras iniciais, variando entre 62,93 e 214,56 UE/mL. O PQM mostrou redução de mais de 99% da microbiota e de 57% da concentração de endotoxinas. Após o uso da MI foram encontrados microrganismos em 54% dos casos, a concentração de endotoxina aumentou consideravelmente comparada ao primeiro PQM. Não houve diferenças estatísticas entre os MI testados. Os antimicrobianos mais eficazes foram a amoxicilina e amoxicilina + ácido clavulânico. Fusobacterium sp. mostraram certa resistência a eritromicina e clindamicina. Em conclusão, os resultados microbiológicos sugerem que o PQM foi responsável pela maior redução na quantidade de microrganismos e endotoxinas. A amoxicilina ou amoxilina + ácido clavulânico devem ser as medicações de primeira escolha quando necessário o emprego de um agente antimicrobiano via sistêmica, para pacientes alérgios aos beta-lactâmicos a azitromicina seria uma alternativa / Abstract: The aim of the present study was to evaluate in vivo the microbial and endotoxins load after chemo-mechanical preparation (CMP) and after the use of intracanal medications (ICM), it was also aimed to evaluate in vitro the antimicrobial suscetibility from some microrganisms isolated. Samples were colected of single root teeth containing necrotic pulp tissue and periapical bone lesions, in several stage of the endodontic treatment. Transport medium, culture and incubation in favour of strict anaerobic microorganisms to grow were used. Microorganisms were identifyed by biochemichal tests and endotoxins quantified by Limulus Amebocite lysate (LAL) test. Antimicrobial susceptibility was evaluated by E-test method using the following antibiotics: benzylpenicillin, amoxicillin, amoxicillin + clavulanate, erythromycin, azithromycin, cephaclor, metronidazole, and clindamycin. Results showed that microorganisms were present in all teeth containing necrotic pulp tissue and periapical bone lesion, the colony forming unit (CFU/mL) ranged from 4x105 to 2.6x106. The microorganisms most frequent isolated were Propionibacterium acnes, Gemella morbillorum, Actinomyces naeslundii and Eubacterium lentum. Endotoxin was also present in all initial samples, ranging from 62.93 to 214.56 UE/mL. Post CMP samples showed more than 99% reduction of cultivable microbiota and 57% (mean value) of endotoxin concentration. After 7 days of ICM, residual microorganisms were still found in 54.1% of the cases and the endotoxin concentration increased considerably compared to the first CMP. No statistically significant differences among the intracanal medicament groups were found. In conclusion, the microbiological results sugested that CMP was responsible for the majority of the microbial and endotoxin reduction. Amoxicilin and amoxicilin plus clavulanic acid were the best antimicrobial agents, as all bacterial strains tested were susceptible to their action. Some Fusobacterium spp. showed to be resistant to eritromycin. In conclusion, the microbiologic results suggest that chemo-mechanical preparation was responsable for mainly microbial and endotoxin reduction. Amoxicilin and amoxicilin puls clavulanic acid are the first choice when required to use a systemic antimicrobial agent, as alternative to alergic patients the azitromicin is an option / Doutorado / Endodontia / Doutor em Clínica Odontológica

M.Sc. / Lipopolysaccharides (LPS) are ubiquitous, indispensable components of the cell surface of Gram-negative bacteria that have diverse roles in bacterial pathogenesis of plants. LPS as pathogen-associated molecular pattern (PAMP) molecules can be recognized by plants to directly trigger some defense—related responses. LPS can also alter the response of plants to subsequent bacterial inoculation; these delayed effects include alterations in the expression patterns of genes coding for some pathogenesis related (PR) proteins, promotion of the synthesis of the antimicrobial conjugates, and prevention of the hypersensitive reaction caused by avirulent bacteria. Prevention of the response may allow expression of resistance in the absence of catastrophic tissue damage. LPS from Burkholderia cepacia (LPSB. cep.) have been found to trigger a strong response in plants resulting in the activation of genes coding for some pathogenesis related proteins, receptor-like kinases and resistance (R) proteins. LPS are tripartite amphipathic molecules, consisting of a Lipid A moiety that is embedded in the outer leaflet of the phospholipids/protein bilayer, a core oligosaccharide, and a polysaccharide consisting of repeating units (0-Antigen/O-side chain). Typically the Lipid A consists of a bisphosphorylated glucosamine disaccharide which is substituted by amide- and ester-bound fatty acids and / or acyloxyacyl groups. The core region, a non-repetitive oligosaccharide, is usually connected to the Lipid A part via one 3-deoxy-D-manno-oct-2-ulosonic (Kdo) residue. The core is attached in turn to the 0- Antigen that consists in most cases of a repetitive polysaccharide and that represents the major part of LPS. The bond between the Lipid A section and the Kdo residue of the core is labile under mild acid hydroysis conditions; and this allows for the fractionation of the LPS molecule into a Lipid A part and an 0-Antigen part, attached to the core. Thus far the eliciting (active) parts of LPSB. cep. have not yet been identified. In general, it is known that the Lipid A is more conserved from one organism to another as compared to the 0-Antigen. In animals, Lipid A is believed to be the active part as it was found to elicit some defense-related responses. In plants, Lipid A was also found to trigger defense responses. Several structures of the 0-Antigens from different bacteria have been characterised, but their biological activities have not yet been investigated in detail.

Because plants lack a circulating adaptive immune system, they have evolved multicomponent defense mechanisms to protect themselves against pathogen attack. These defense mechanisms/responses are either constitutively active in the plant, or they are inducible by pathogens. Understanding of the plant response to pathogen attack has advanced rapidly in recent years. Bacterial and fungal pathogenicity factors have been isolated, and mechanisms that are utilized by the plant to recognize the pathogen and initiate a plethora of defense mechanisms have been identified. In contrast to the well-documented effects of LPS on mammalian cells, the effects of LPS on plant cells have been far less studied. The present study focused on the involvement of lipopolysaccharides (LPS) isolated from the outer cell wall of the Gram-negative bacteria, Burkholderia cepacia (strain ASP B 2D), and yeast elicitor (YE, a cell wall preparation from Saccharomyces cerevisiae) on the molecular mechanisms and components involved in signal transduction and defense-related responses in suspension cultured cells derived from tobacco plants (Nicotiana tabacum cv. Samsun). LPS was extracted, analyzed by denaturing electrophoresis and characterized with regard to 2-keto-3-deoxyoctonate (KDO) content, carbohydrate content, and protein content. The purified LPS and YE were found to trigger defense- and resistance-related responses in the tobacco cells. These responses included a rapid influx of Ca2+ into the cytoplasm of transgenic aequorin-transformed tobacco cells, the production of reactive oxygen species (ROS) during the oxidative burst, alkalinization of the extracellular culture medium of the cells, and changes in the protein phosphorylation patterns of the cells. Time- and concentration-dependent studies for the induction of perception and signal transduction-related responses by YE and LPS indicated that 100 µg.ml-1 of either elicitor was sufficient to induce significant responses in the cells. YE and LPS both induced a rapid transient increase in cytosolic Ca2+ levels, returning to basal levels after seconds, followed by a second, larger and long-term increase in cytosolic Ca2+. The YE-induced cytosolic Ca2+ influx was 7.5 fold higher than that of LPS. Luminol-dependent chemiluminescence measurements of hydrogen peroxide (H2O2) produced during the YE- and LPS-induced oxidative burst reactions indicated 3.5 fold higher levels of H2O2 induced by YE than that induced by LPS. Total inhibition of H2O2 production by YE- and LPS-induced cells was observed upon treatment of the cells with the H2O2-degrading enzyme, catalase. ROS production was also analyzed by the H2DCF-DA-derived fluorescence assay. The degree of ROS production by YE-treated cells was larger than that of cells treated with LPS, suggesting that YE is a more potent inducer of plant defense responses than LPS. Categorization of the origins of the oxidative bursts, induced by YE and LPS, by the addition of a ROS scavenger (NAC), inhibitors of ROS production (DPI and DDC) and a nitric oxide scavenger (PTIO) indicated that YE and LPS induced different quantities of the same ROS species. The induced ROS included O2-·, H2O2 and perhaps other ROS species as well. In addition, both YE and LPS induced a remarkable burst of nitric oxide (NO), as determined by the 97% and 95% respective inhibitions of the H2DCF-DA-derived fluorescence by the nitric oxide scavenger PTIO. Alkalinization of the extracellular culture medium of the tobacco cells was observed after treatment of the cells with YE and LPS. Both of these elicitors induced a significant increase in extracellular pH from resting pH values of 5.7 to pH 6.3 by YE, and 6.0 by LPS. Notably, the YE-induced response returned to near basal pH levels after 50 min, while the LPS-induced response showed no signs of declining and fluctuated around pH 5.9 for the duration of the experiment. YE and LPS both induced the hyperphosphorylation of two distinct proteins with approximate molecular masses of 28 kDa and 2 kDa. Changes in the pattern of the [32P]-radiolabeled proteins pp28 became visible after 20 min of YE-elicitation and 30 min of LPS-elicitation and changes in pp2 phosphorylation became visible after 20 min treatment of the cells with both elicitors. Addition of the protein kinase inhibitor, staurosporine, to the cells followed by subsequent elicitation by YE or LPS, resulted in inhibition or abolishment of the elicitor-induced responses during the oxidative burst, extracellular alkalinization, and protein phosphorylation. In contrast, the addition of the protein phosphatase inhibitor, calyculin A, was found to mimic elicitor action in several aspects, including extracellular alkalinization, the oxidative burst and protein phosphorylation, even in the absence of elicitors or any other stimulus. Thus, a fine balance between the actions of certain protein kinases and protein phosphatases is an essential component of signal transduction during YE and LPS elicitation of tobacco cells but the identification and characterization of the staurosporine-sensitive protein kinases and their substrates are necessary to gain a better understanding of the chemosensory perception and signal transduction of the YE and LPS elicitor signals in plant cells. Moreover, the question of whether these perception and transduction mechanisms are connected with a reduced activity of a protein phosphatase, or with the increased activity of a protein kinase, or even a combination of both, remains to be elucidated. / Prof. I.A. Dubery

Contexte : Le cancer du poumon est le cancer le plus associé aux expositions professionnelles. Bien que l’amiante soit sans doute le facteur étiologique professionnel le plus connu, plusieurs éléments suggèrent également l’implication de facteurs modifiables d’origine environnementale ou professionnelle, beaucoup plus rarement étudiés, parmi lesquels les agents biologiques. Si les endotoxines - toxines situées dans la membrane externe des bacilles Gram négatif - sont fréquemment associées à une réduction du risque de cancer du poumon, les infections à papillomavirus humains sont suspectées d’augmenter ce risque. Les résultats des études épidémiologiques sur le sujet sont divergents et sont régulièrement limités par de nombreuses faiblesses méthodologiques incluant entre autres la non prise en compte du tabagisme et de l’exposition à l’amiante.Objectifs : L’objectif de cette thèse est d’étudier le rôle des agents biologiques présents sur les lieux de travail dans la survenue des cancers broncho-pulmonaires. Plus spécifiquement, les objectifs de ce travail sont : (1) d’étudier le rôle de l’exposition professionnelle aux endotoxines sur le risque de cancer du poumon, d’évaluer les aspects de la relation dose-effet ainsi que les interactions possibles avec les antécédents des maladies respiratoires; (2) de s’intéresser plus particulièrement à l’industrie textile, source de fortes expositions aux endotoxines, en étudiant le risque de cancer du poumon associé à l’exposition professionnelle aux poussières textiles ; (3) d’étudier le risque de cancer du poumon dans l’industrie de la viande en testant, notamment l’hypothèse d’une exposition à un agent viral.Population et méthodes : Ce travail s’est appuyé sur les données de l’étude cas-témoins en population générale ICARE. Les cas de cancer du poumon ont été identifiés dans 10 départements français abritant un registre général de cancer. Au total, 2276 cas de cancer du poumon et 2780 témoins hommes ont été inclus ainsi que 650 cas de cancers du poumon et 775 témoins femmes. Les descriptions détaillées de l’histoire professionnelle complète recueillies par des questionnaires standardisés ont permis de coder les professions et les secteurs d’activités de chaque emploi selon les classifications CITP 1968 et NAF 2000.Résultats : Nous avons trouvé une association inverse entre les expositions professionnelles aux endotoxines et le risque de cancer du poumon particulièrement plus marquée chez les travailleurs de l’élevage (tous types) et de la collecte et traitements des déchets. Les odds ratios de cancer du poumon diminuent avec la durée et l’indice cumulé d’exposition aux endotoxines. Nos résultats ne soutiennent pas l'existence d'une forte association entre l'exposition professionnelle aux poussières textiles et le cancer du poumon, néanmoins, ils montrent une diminution significative de 30% du risque de cancer du poumon chez les travailleurs du coton avec OR= 0,7 ; IC 95% [0,5-0,9]. Par ailleurs, nous observons une association positive significative avec le risque de cancer du poumon chez les travailleurs de l’industrie de la viande (OR= 1,46 [1,0-2,1]). Cependant, les antécédents de verrues de la main ne semblent pas avoir d’effet modificateur dans l’association entre le travail dans l’industrie de la viande et le risque de cancer du poumon.Conclusion : Nos résultats soutiennent le rôle important des expositions professionnelles comme déterminants du risque de cancer du poumon. Ils confirment les associations inverses entre les expositions aux endotoxines et le risque de cancer du poumon suggérant fortement l’hypothèse d’un effet anti-tumoral des endotoxines vis-à-vis du poumon. Par ailleurs, ils semblent indiquer que le travail dans l’industrie de la viande est une situation d’exposition à risque de cancer du poumon sans toutefois conclure formellement quant à l’implication des infections à papillomavirus humains. / Background: Lung cancer is the most common cancer associated with occupational exposures. Although asbestos is the best known occupational etiologic factor, several hypotheses suggest the involvement of some environmental or occupational modifiable factors, much more rarely studied, including biological agents. Endotoxins-toxins being part of the outer membrane of Gram-negative bacilli- are commonly associated with reduced risk of lung cancer, while human papillomavirus infections are suspected of increasing the risk. However, the majority of published studies had small numbers of cases and some methodological issues such as inadequate adjustment for tobacco smoking and occupational exposure to asbestos.Objectives: The aim of the present work is to study the role of biological agents found in the workplace on lung cancer risk and more specifically: (1) to investigate the role of occupational exposure to endotoxins on lung cancer risk, to assess dose-response relationship aspects and possible interactions with history of respiratory disease; (2) to focus on the textile industry, which is a source of high exposure to endotoxins, by examining the risk of lung cancer associated with occupational exposure to textile dust; (3) to study the risk of lung cancer in the meat industry by testing the hypothesis of an exposure to a viral agent.Population and methods: This work was based on a large population-based case-control study, ICARE. Cases and controls were recruited from 10 French departments. Incident cases were identified through French cancer registries. A total of 2,926 lung cancer cases (2,276 men and 650 women) and 3,555 controls (2,780 men and 775 women) were included. Detailed information on lifetime occupation was collected through standardized questionnaires. Jobs and sectors were coded according to the ISCO 1968 and the NAF 2000 classifications.Results: Our results showed an inverse association between exposure to endotoxins and lung cancer risk particularly among livestock (all types) and waste collection and treatment workers. Negative trends were shown with duration and cumulative exposure. Furthermore, our findings do not the existence of strong association between occupational exposure to textile dust and lung cancer. A decreased risk was suggested for distant exposures and for work with cotton fibres (OR = 0.7; 95% CI [0.5-0.9]). On the other hand, we observe a significantly increased risk among meat workers OR = 1.46 [1.1-2.1]). However, the history of hand warts does not seem to have any modifying effect on the association between work in the meat industry and the risk of lung cancer.Conclusion: Our findings support the important role of occupational exposures as a determinant of lung cancer risk. This confirms the inverse associations between endotoxin exposures and the risk of lung cancer strongly suggesting an anti-tumor effect of endotoxins towards the lung. This also seems to indicate that working in the meat industry is a risky situation for lung cancer without, however, formally concluding about the involvement of human papillomavirus infections.

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Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vi, 52 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 42-49).

Endotoxins are a constituent of the lipopolysaccharide (LPS) complexes present in the outer layer of the cell wall of most Gram-negative bacteria and some cyanobacteria. The ingestion by a typical adult of amounts exceeding 1,000 endotoxin units (EUs) can cause fever, diarrhoea, vomiting, acute respiratory illnesses, and lung inflammation. In contrast, much smaller doses may lead to protective immunity against allergic diseases. / Endotoxins can be released in the air as well as in the water; previous studies have mainly focused on airborne endotoxins. Although many studies on endotoxins in raw and treated drinking waters have been performed, few have assessed seasonal variations and none have been conducted in Eastern Canada. Furthermore, a clear understanding of removal of endotoxins by various water treatment processes is still required. / Two methods to measure the concentrations of endotoxin were used and compared, the Limulus Amebocyte Lysate test (LAL) and the recombinant Factor C test (rFC). Raw water samples were taken from various drinking water sources around the Island of Montreal. The effects of free chlorine, UV radiation, and ozone were studied in batch experiments on filtered water samples via typical dosages and fluences used in drinking water treatment facilities. Residual concentrations for free chlorine were 0.8 and 1.6 mg/L; ozone doses were 0.5 and 1 mg/L; UV fluences were 40 and 100 mWs/cm2. Detention times of 20 and 60 minutes were tested for chlorine and 5 and 20 minutes for ozone. Grab sampling from three drinking water treatment plants in the Montreal area was performed during the months of June and late August/September 2006 and January 2007. Processes at these plants include coagulation and flocculation, sand filtration, ozonation and disinfection by chlorine. To test the variation in endotoxin concentrations during a sand filter cycle, samples were withdrawn directly from a filter in one of the treatment plants studied. The filtration cycle, from one backwash to the next one, lasts 72 h. Samples were collected immediately before the backwash, at the beginning and at the end of the ripening period, at the beginning of the filtration cycle and 48 h later, which corresponds to a half cycle period. / Of the two endotoxin detection methods used, LAL consistently gave slightly higher values compared to rFC; rFC also required more expensive hardware, but the method was less tedious and reagent costs were lower. Results presented, unless otherwise stated, were obtained with the rFC method. Endotoxin levels decreased in raw water samples between June and September. Concentrations ranged from 20 to 30 EU/mL in June, and decreased to 10 to 14 EU/mL in August and beyond. For the disinfection processes, the UV and free chlorine doses tested had little or no effect on the endotoxin concentrations, but ozone reduced the concentrations by up to 75%. Sand filtration and flocculation showed significant endotoxin removal efficiencies (50--60%). Levels remained around 5 EU/mL throughout the remaining treatment processes regardless of the influent concentration. Hence, endotoxin inactivation by free chlorine and UV does not occur with typical doses used in drinking water treatment plants; in contrast, flocculation and sand filtration, as well as ozonation, are much more effective.