Este estudo clínico analisou os efeitos dos procedimentos endodônticos e da irrigação ultrassônica passiva (PUI) em bactérias e endotoxinas de canais radiculares. Cinquenta pacientes com dentes com periodontite apical primária foram divididos de forma randomizada em dois grupos: PUI (n=25) e irrigação convencional (IC) (n=25). O preparo químico-cirúrgico (PQC) foi realizado com instrumentos reciprocantes, utilizando-se NaOCl 2,5% durante o preparo; e EDTA 17%, para remoção do magma dentinário. Os canais radiculares foram preenchidos com pasta de hidróxido de cálcio por 14 dias e obturados. Foram realizadas coletas microbiológicas dos canais antes (S1) e após o PQC (S2), após os protocolos de irrigação (S3), após a medicação intracanal (S4) e após a reinstrumentação dos canais (S5). Durante o processamento das amostras, as coletas de 5 casos foram perdidas por fatores diversos. As amostras foram analisadas por PCR quantitativo para detecção e quantificação de bactérias e pelo teste turbidimétrico de LAL para detecção e determinação do nível de endotoxinas. Bactérias e endotoxinas foram observadas em 100% das amostras iniciais coletadas. Em ambos os grupos, houve diminuição significativa na concentração de endotoxinas entre uma etapa do tratamento e a etapa posterior (p<0,05). O mesmo foi observado quanto ao número de bactérias, exceto entre a remoção da medicação intracanal e a reinstrumentação antes da obturação (S5). A análise intergrupos demonstrou que, com relação às endotoxinas, não foram observadas diferenças significativas entre os grupos (p>0,05). Com relação à redução bacteriana, a PUI foi capaz de reduzir significativamente mais o número de bactérias do que a IC (p<0,05). A análise qualitativa dos resultados demonstrou que nenhuma das técnicas sobressaiu-se sobre a outra em relação à frequência de casos com leituras positivas, tanto para bactérias quanto para endotoxinas. Concluiu-se que a PUI foi mais eficaz do que a IC em reduzir o número de bactérias, mas não a quantidade de endotoxinas do canal radicular. Além disso, cada etapa do tratamento endodôntico foi capaz de reduzir tanto o número de bactérias quanto a quantidade de endotoxinas. / The aim of this clinical study was to compare the effects of passive ultrasonic irrigation (PUI) and intracanal medication with calcium hydroxide in bacteria and endotoxins from root canal. Fifty teeth with apical periodontitis were randomly divided into two groups according to the irrigation protocol: PUI (n = 25) and conventional irrigation (CI) (n = 25). The root canal preparation of all the teeth was carried out with reciprocating files and 2.5% NaOCl during preparation; and 17% EDTA for smear layer removal. The root canals were medicated with calcium hydroxide for 14 days. Microbiological sampling were performed before (S1) and after the preparation (S2) after irrigation protocols (S3), and after intracanal medication (S4 and S5). During the processing of the samples, five cases were lost for several factors. The samples were analyzed by real time PCR, for the detection and quantification of bacteria, and the turbidimetric LAL assay, for the detection and analysis of the endotoxin levels. Bacteria and endotoxins were observed in 100% of the initial samples. In both groups, there was a significant decrease in the concentration of endotoxins between one step and the subsequent step of treatment (p < 0.05). The same was observed for the number of bacteria, with the exception of the reinstrumentation after the removal of the medication (S5). The intergroup analysis showed no significant differences between groups in endotoxin reduction ( p > 0.05 ). PUI was able to reduce the number of bacteria significantly better than CI (p < 0.05). No significant statistical difference was observed between groups regarding the occurrence of cases wielding positive results for bacteria or endotoxin. It was concluded that PUI was more effective than CI in reducing the number of bacteria but not the amount of endotoxin in the root canal. Furthermore, each step of the endodontic therapy was effective in reducing both the number of bacteria as the amount of endotoxin.

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Oceanografia Física, Química e Geológica, Instituto de Oceanografia, 2007. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2013-02-18T15:57:58Z No. of bitstreams: 1 2007_fabiana_gutierrez.pdf: 651273 bytes, checksum: 521f5497a65fcefafef6ef4795036615 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2013-06-12T16:59:29Z (GMT) No. of bitstreams: 1 2007_fabiana_gutierrez.pdf: 651273 bytes, checksum: 521f5497a65fcefafef6ef4795036615 (MD5) / Made available in DSpace on 2013-06-12T16:59:29Z (GMT). No. of bitstreams: 1 2007_fabiana_gutierrez.pdf: 651273 bytes, checksum: 521f5497a65fcefafef6ef4795036615 (MD5) Previous issue date: 2007 / Florações de cianobactérias têm sido freqüentemente encontradas nas águas do estuário da Lagoa dos Patos (RS). Um das principais cianobactérias de ocorrência local é o gênero Microcystis. As células de Microcystis têm tolerância a baixas salinidades, porém com o aumento abrupto da salinidade, as células podem sofrer lise. Muitas vezes, em função da hidrodinâmica local, essas florações de cianobactérias podem atingir a região da Praia do Cassino, possibilitando o contato com banhistas. Devido à natureza Gram-negativa da composição da parede celular das cianobactérias, esse contato com as células pode resultar em na ocorrência de casos de irritação epicutânea e reações alérgicas. O agente causador é o lipídeo A (endotoxina), encontrado no lipopolissacarídeo (LPS) da membrana externa das cianobactérias. Com o objetivo de detectar concentrações ambientais de LPS das florações de cianobactérias foi utilizado o método cromogênico cinético de ponto final do teste do Lisado do Amebócito de Limulus polyphemus (teste LAL). O teste LAL foi realizado em amostras de água de superfície coletadas nas regiões de São Lourenço do Sul, Praia do Laranjal (Pelotas), Museu Oceanográfico da FURG, Yacht Club do Rio Grande e Praia do Cassino (Rio Grande), entre os meses de dezembro de 2005 e março de 2006. Nestas amostras também foram estimados a abundância celular dos principais grupos do fitoplâncton, a concentração de clorofila-a, e medida a salinidade local no momento da coleta. Para avaliar a possível interferência dos sais nos resultados do teste LAL, também foram realizados cultivos da cianobactéria Microcystis crescendo em diferentes salinidades, onde também foram estimados a abundância celular, e as concentrações de clorofila-a, LPS e microcistinas. Os ajustes metodológicos realizados no teste LAL durante o trabalho resultaram em uma sensibilidade para sua aplicação nas amostras ambientais. As maiores concentrações de endotoxinas detectadas nas amostras ambientais (109,5 EU.mL-1, 71,8 EU.mL-1 e 93,7 EU.mL-1) se correlacionaram positivamente com as maiores abundâncias celulares (aproximadamente 600.000 células.mL-1, 400.000 células.mL-1 e 300.000 células.mL-1, respectivamente), todas com a presença da cianobactéria Microcystis. / Cyanobacterial blooms have been frequently observed in the waters of the Patos Lagoon estuary (RS). One of the main cyanobacterium of local occurrence is the genus Microcystis. The Microcystis cells present some tolerance to low salinity, but with the rapid salt increase the cells may suffer breakdown. Usually due to the local hydrodynamics these blooms may reach the recreational waters of the Cassino Beach, exposing swimming bathers to its contact. Due to the Gram-negative nature of the cyanobacterial cell membrane, this contact with the cells may result in case occurrences of epicutaneous irritation and allergic reactions. The causing agent is the lipid A (endotoxin), found on the lipopolysaccharide (LPS) of the external membrane of cyanobacteria. With the objective to detect environmental concentrations of LPS from cyanobacterial blooms, it was used the kinetic chromogenic endpoint method of the Limulus polyphemus Amebocyte Lisate test (LAL test). The LAL test was performed in surface water samples collected from the regions of São Lourenço do Sul, Laranjal Beach (Pelotas), FURG’s Oceanographic Museum, Yacht Club Rio Grande, and Cassino Beach (Rio Grande) betwen December 2005 and March 2006. In these samples it was also estimated the cellular abundance of the main phytoplanktonic groups, the chlorophyll-a concentration, and measured the local salinity. To evaluate the possible interference of salinity in the LAL test results, it was also cultivated the cyanobacterium Microcystis growing in different salinities. From these cultures it was estimate the cellular abundance, and chlorophyll-a, LPS and microcystin concentrations.. The methodological adjustments performed in the LAL test during this work resulted in a sensitivity for its application in environmental samples. The highest endotoxin concentrations detected in the environmental samples (109.5 EU.mL-1, 71.8 EU.mL-1 and 93.7 EU.mL-1) have positively correlated with the highest cell abundances (approximately 600,000 cells.mL-1, 400,000 cells.mL-1 and 300,000 cells.mL-1, respectively), all of them with the presence of the cyanobacteria Microcystis.

While protein therapeutics are indispensable in the treatment of a variety of diseases, including cancer, rheumatoid arthritis, and diabetes, key limitations including short half-lives, high immunogenicity, protein instability, and centralized production complicate long-term use and on-demand production. Site-specific polymer conjugation provides a method for mitigating these challenges while minimizing negative impacts on protein activity. However, the location-dependent effects of polymer conjugation are not well understood. Cell-free protein synthesis provides direct access to the synthesis environment and rapid synthesis times, enabling rapid evaluation of multiple conjugation sites on a target protein. Here, work is presented towards developing cell-free protein synthesis as a platform for both design and on-demand production of next generation polymer-protein therapeutics, including (1) eliminating endotoxin contamination in cell-free reagents for simplified therapeutic preparation, (2) improving shelf-stability of cell-free reagents via lyophilization for on-demand production, (3) coupling coarse-grain simulation with high-throughput cell-free protein synthesis to enable rapid identification of optimal polymer conjugation sites, and (4) optimizing cell-free protein synthesis for production of therapeutic proteins

Therapeutic proteins have become a cornerstone of modern medicine since the FDA approval of recombinant human insulin in 1982. Likewise, biosensors transform chemical detection and disease diagnostics by identifying biomarkers, chemical contaminants, and infective agents. Long-standing methods for creating therapeutics and biosensors employ whole cells such as Escherichia coli (E. coli). Alternatively, cell-free protein synthesis (CFPS) employs the enzymatic reactions necessary for protein production and biosensing within a cell, but in an engineered reactor environment facilitating unprecedented access to and control over biochemical machinery, preservation by cryodesiccation for portable deployment, and functionality in cytotoxic applications. This dissertation reports advances in an E. coli CFPS production platform toward creating therapeutic proteins by this means. First, an endotoxin-free CFPS platform is created by optimizing fermentation and cell-extract harvest of an endotoxin-free E. coli strain. Next, liquid cell growth culture media is specially formulated to change chemical composition during cell culture and provide a streamlined method for producing high-yielding, endotoxin-free E. coli CFPS. Then, novel CFPS bioreactor formats are mathematically validated and developed which employ "hydrofoam" and oxygen to increase therapeutic protein production yield. Additionally, advances are reported in CFPS biosensing technology. First, a chimeric fusion protein incorporating the ligand binding domain of the human estrogen receptor is expressed in CFPS to detect estrogenic chemicals in the presence of human blood and urine. Next, the molecular mechanism of this protein construct is elucidated and the assay readout is optimized with mathematical simulations and CFPS. Then, CFPS is metabolically engineered to create a biosensor of L-glutamine, the most abundant amino acid in the body. Finally, this dissertation reports the development of a synergistic platform for potentially treating Acute Lymphoblastic Leukemia wherein CFPS is engineered to both produce the therapeutic protein crisantaspase and assess its activity in the presence of human serum for improved, potentially even personalized treatment of the disease. It is anticipated that the advances reported herein will contribute to the utility of in vitro or cell-free protein synthesis for therapeutic and diagnostic applications.

Over the last decades, there has been much attention on air quality, especially in urban environments. A significant factor effecting the air quality in the urban environment is airborne particulate matter (PM). Long-term exposure to PM causes increased risk for heart disease, decreased lung function, exacerbation of asthma, and lung cancer. Therefore, many countries have implemented exposure limits to the concentration of ambient PM in the urban environments. The toxicity of PM is dependent on several factors such as chemical composition, shape, adsorbed materials, and particle sizes (usually divided in ultrafine, fine and coarse particles). However, the relationship between different PM properties and developing health hazards are not clear. Therefore, further studies to investigate different properties of PM may contribute to understanding the influence of PM on human health.  In the present work, different novel methods to investigate sampled airborne PM and to investigate potential health effects have been used to increase the knowledge regarding street and wear particles. In study I, a sampling plan involving collecting one filter from Tapered Element Oscillating Microbalance (TEOM) monitoring stations used for Environmental monitoring each month for 20 months were developed. Particles on the filters were extracted into a suspension. TEOM particles were then studied with respect to constituents and variables that reflect their toxicity. The constituent and toxicity was found to be spatial and seasonal dependent. As a follow-up, in study II, TEOM filters from three geographical different cities were collected once a month for a year. The variation in particle mass measured with TEOM monitor, cadmium and lead contents, as well as endotoxin levels between locations and time points over the year was studied. The correlation between studied variables and biological effect was investigated. The results show that the concentration of metals and endotoxin in TEOM particles have no relationship to particle mass, while endotoxin levels coincided with pro-inflammatory response. These studies show that results from analyzing different variables on obtained particles from TEOM filters in combination with information about the ambient particle concentration, could be helpful in the evaluation of differences in the risk of breathing air at various locations.  The dominant road traffic particle sources are wear particles from the road and tyre interface, and from vehicle brake pads. The particle concentrations are highest in cities with high traffic amounts and a high frequency of braking. There are a few cell studies that have investigated the toxicological and biological effect of these wear particles, but there is a lack of knowledge regarding their effect on tissue level. Furthermore, the knowledge about importance of rock materials in pavement is deficient. To mitigate these knowledge gaps, the effect of different wear particles from pavement and brake pad were tested using a model with isolated perfused rat lungs in study III. The wear particles from the pavement showed a significant decrease of tidal volume compared to unexposed controls. The largest effect were found with quartzite stone material. Wear particles from brakes instead showed a larger effect on released proinflammatory cytokines. The study shows that the toxic effect on lungs exposed to airborne particles can be investigated using repetitive measurements of tidal volume. Furthermore, the study shows that the choice of rock material in road pavements has the potential to affect the toxicity of road wear particles. This should be considered in environments where the concentrations and exposures are high. The brake particles showed a different effect than stone particles, indicating the need to differentiate wear particles from different sources in relation to health effects. In summary, the present work have investigated different aspects of airborne particles collected from streets as well as generated wear particles. These indicate different important aspects of the particles that may be of importance to better understand their health effects.

Heterotrimeric Gi proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS-induced, heat-killed SA-induced and heat-killed GBS-induced cytokine and chemokine production in peritoneal macrophages from wild-type (WT), Gαi2-/- or Gαi1/3-/- mice were investigated. LPS induced production of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10 and interferon-γ-inducible protein-10 (IP-10); SA induced TNF-α, and IL-1β production; and GBS induced TNF-α, IL-6, IL-1β, macrophage inflammatory protein-1α (MIP-1α) and keratinocyte chemoattract (KC) production were all decreased (P < 0.05) in Gαi2-/- or Gαi1/3-/- mice compared with WT mice. In contrast to the role of Gi proteins as a positive regulator of mediators, LPS-induced production of MIP-1α and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in macrophages from Gαi1/3-/- mice, and SA-induced MIP-1α production was increased in both groups of Gαi protein-depleted mice. LPS-induced production of KC and IL-1β, SA-induced production of GM-CSF, KC and IP-10, and GBS-induced production of IL-10, GM-CSF and IP-10 were unchanged in macrophages from Gαi2-/- or Gαi1/3-/- mice compared with WT mice. These data suggest that Gi2 and Gi1/3 proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram-positive microbial stimuli.

Asthma is the leading chronic illness in children in the United States. Since children in the U.S. spend a majority of their time indoors there is an increased need to understand key sources of daily asthma triggers in the home. Bacterial endotoxin, dust mite allergens and β-D-glucan have been shown to be potent inducers of asthma attacks, and high levels of these allergens in homes can trigger attacks in those with asthma. We aim to better understand the risks to those with asthma that might be associated with evaporative cooler (EC) use in low-income homes. ECs are often promoted because of their low energy consumption and decreased environmental impact compared to central air conditioning (AC). Because of their lower cost, ECs are more widely used in low-income homes. ECs use evaporation to cool the air, which leads to higher indoor relative humidity. This may create an ecological niche for house dust mites in semi-arid climates where they are normally absent. EC sump water also provides an ideal environment for bacteria and fungi to grow, possibly resulting in EC loading the air with more potential asthma triggers than central air conditioning. We sampled low-income homes around Utah county with central air and evaporative cooling and tested them for the presence of dust mite allergens, β-D-glucan and endotoxin. There were significantly higher levels of endotoxins and β-(1→3)-D-glucans in the EC homes compared to the AC homes, with increased odds of dust mite allergen prevalence but not at clinically significant levels. These findings suggest that in semi-arid environments, endotoxin and β-(1→3)-D-glucan levels in homes with evaporative coolers are more elevated than dust mite allergens.

No description available.

We investigated the hypothesis that exposure to inhaled diesel exhaust PM can alter the gut microbiome and intestinal integrity, thereby promoting systemic inflammatory response and early CVD risk, which are exacerbated by HF diet. Furthermore, we investigated whether the observed exposure and diet-mediated outcomes could be mitigated through probiotic treatment. We performed an exposure study on C57Bl/6 male mice, placed on either a low fat (LF) diet or a high-fat (HF) diet, and exposed via oropharyngeal aspiration to 35 μg diesel exhaust particles (DEP) suspended in 35 μl of sterile saline or sterile saline controls (CON) twice a week for four weeks. A subset of mice on HF diet were dosed with 0.3 g/day (PRO, ~7.5x108 CFU/day) of probiotic Ecologic® Barrier 849 (Winclove Probiotics) in drinking water during the course of the study. For our first aim, we investigated the alterations in the gut microbiome, measured circulating cytokines and lipopolysaccharide (LPS), and measured CVD biomarkers in the heart. Our results revealed that exposure to inhaled DEP results in gut dysbiosis characterized by expansion of the phyla Verrucomicrobia and Proteobacteria and reduction in Actinobacteria, which was exacerbated by HF diet. Probiotics mitigated the DEP-mediated expansion of Proteobacteria and re-established Actinobacteria in the intestine of HF animals. Furthermore, we determined that exposure to inhaled DEP increases systemic LPS and inflammatory markers IL-1α, IL-3, G-CSF, and TNF-α. Furthermore, we found that inhaled DEP exposure results in increased CVD biomarkers sICAM-1, sP-selectin, and thrombomodulin in the heart. Probiotic treatment was effective in attenuating LPS, inflammatory responses, and CVD biomarkers in HF animals, validating the involvement of the microbiome in mediating inhaled DEP-mediated responses. Considering the effects we observed in the microbiota and systemically of the HF and probiotic treatment animals, we investigated the effects of inhaled DEP on intestinal integrity and inflammation in HF ± PRO animals. Our results showed that inhaled DEP in conjunction with HF diet promotes increased goblet cell and mucin 2 expression, tight junction (TJ) proteins (claudin-3, occludin, and zonula occludens (ZO)-1) expression, matrix metalloproteinase (MMP)-9, toll-like receptor (TLR)-4, and decreased TNF-α and IL-10. Moreover, we found that probiotics promoted intestinal immune response following inhaled DEP exposure characterized by an increase in TNF-α and IL-10 and showed variable expression of TJs in the intestine. In conclusion, we found that inhaled DEP exposure results in changes in gut microbial profiles, altered intestinal integrity, systemic inflammation, and increased CVD biomarkers, which are exacerbated by HF diet. The use of probiotics in this study proved to be pivotal in understanding the microbiome's influences on the regulation of intestinal integrity, intestinal inflammation, systemic inflammation, and cardiovascular system following inhaled DEP exposure with HF diet.

The quality of marine bathing water is currently assessed by monitoring the levels of faecal indicator bacteria. Among other drawbacks, results are retrospective using the traditional culture based methods. A rapid method is thus needed as an early warning to bathers for bacterial contamination in marine bathing waters. Total lipopolysaccharide (LPS) was chosen here as a potential general biomarker for bacterial contamination. Levels of total LPS, measured using a Kinetic QCL™ Limulus Amebocyte Lysate (LAL) assay, highly correlated with enumerated Escherichia coli and Bacteroides species. Levels of LPS in excess of 50 EU mL-1 were found to equate with water that was unsuitable for bathing under the current European Union regulations. Results showed that monitoring the levels of total LPS has a potential applicability as a rapid method for screening the quality of marine bathing water. More importantly, the LAL assay overcome the retrospective results when using culture based assessment since the LAL assay takes less than 30 minutes. Although false positive events were not detected, the occurrence of a false positive has been hypothesised, hence a more specific faecal biomarker was also investigated. LPS of five Bacteroides species (B. fragilis, B. caccae, B. ovatus, B. xylanisolvens and B. finegoldii) isolated from marine bathing waters samples were successfully profiled and showed high similarity between isolates in LPS gel electrophoresis banding pattern. Similar results were shown when investigating the endotoxic activity of Bacteroides species with the Kinetic QCL™ LAL assay. The potential biological relevance of Bacteroides LPS was also investigated in cell culture models indicating that Bacteroides showed similar induction of proinflammatory cytokines (TNF-α, IL-6 and IL-1α) and generally the biological activity was approximately 100 fold less than E. coli LPS. In addition, an ELISA assay was designed for the detection of Bacteroides LPS. Results showed that the Bacteroides LPS has a high potential to be used as a faecal biomarker, however, further work is required to develop a fully functional assay. The potential biological relevance of LPS present in contaminated bathing waters was also investigated in cell culture models. Results showed that there is a significant difference in the production of proinflammatory cytokines in comparison to “clean” bathing waters. Thus, results suggest that the European Directive regulations should be extended to cover the levels of total LPS in bathing waters to assure safety to the users of marine recreational water.