Em países industrializados, as doenças pulmonares são as que mais se destacam quando o assunto é doença ocupacional. Entre os técnicos, cuidadores de animais, médicos e cientistas, as doenças respiratórias e alérgicas a animais de laboratório representam a principal doença ocupacional. Entre os agentes presentes na sujeira orgânica, as endotoxinas são as mais relacionadas às respostas inflamatórias e causadoras de uma série de doenças respiratórias. As endotoxinas, componentes externos das bactérias gram-negativas, são encontradas em várias concentrações em suspensão no ar ou depositadas na poeira do chão, em materiais e equipamentos, no ambiente domiciliar, urbano, rural e em alguns estabelecimentos. É inevitável a exposição à endotoxina, no entanto, o nível de exposição das vias aéreas pode ser muito variado. Alguns estudos mostraram a correlação dos sintomas apresentados pelos trabalhadores de laboratórios ou biotérios com o nível de exposição às endotoxinas. OBJETIVOS: Avaliar a exposição às endotoxinas, presentes na poeira de laboratórios e biotérios e a sua relação com asma, rinite e atopia apresentadas pelos trabalhadores. MÉTODOS: Trata-se de um estudo transversal, realizado na Universidade de São Paulo, campus Ribeirão Preto (USP-RP) e na Universidade Estadual de Campinas (UNICAMP). Foram coletadas amostras de poeira do chão de laboratórios e biotérios que continham rato, camundongo, cobaia, coelho ou hamster; e em laboratórios e salas administrativas que não tinham contato algum com esses animais. As amostras de poeira foram analisadas e a quantidade de endotoxina foi dosada pelo método Limulus amebocyte lysate (LAL). Esta quantidade foi relacionada com variáveis clínicas dos trabalhadores destes locais (sintomas, reatividade brônquica, espirometria e testes alérgicos). RESULTADOS: Foram coletadas amostras de poeira de 145 locais de trabalho. Destes, 74 (51%) da USP-RP e 71 (49%) da UNICAMP. Noventa e dois (63%) locais de trabalho continham animais de laboratório (57 da USP-RP e 35 da UNICAMP) e 53 não os continham (17 da USP-RP e 36 da UNICAMP). Foram utilizados os dados de 751 trabalhadores, 412 formaram o grupo exposto a animais de laboratório e 339 o grupo não exposto. O grupo exposto a animais de laboratório apresentou maior quantidade de endotoxina, 55 ± 79 UE por mg de poeira, quando comparado com o grupo não exposto, 19 ± 27 UE/mg (p < 0,001 pelo teste t de Student). Quando estratificada, a quantidade de endotoxina em elevada e baixa quantidade, a alta concentração (acima de 20,4 UE/mg) de endotoxina se associou ao relato de sibilos nos últimos 12 meses. Ou seja, 27% dos trabalhores expostos a elevadas concentrações relataram sibilos (p < 0,01 pelo teste do qui-quadrado). Porém, a quantidade de endotoxina não se associou com sintomas de rinite, com atopia, com o teste de hiperreatividade brônquica positiva. CONCLUSÃO: A exposição à endotoxina apresenta efeito no sistema respiratório dos trabalhadores mesmo não tendo se associado à asma. A alta concentração de endotoxina se associou com a presença de sibilos, ou seja, os trabalhadores de biotérios e de laboratórios que tem contato direto com animais de laboratório estão mais susceptíveis a apresentarem sibilos ao longo do ano, sendo necessárias medidas de prevenção para estes trabalhadores. / Respiratory diseases are the most common occupational illnesses in industrialized countries and, among them, asthma and allergies are highly prevalent. Respiratory diseases and laboratory animal allergies represent a major occupational illness among technicians, animal caretakers, doctors and scientists whose work requires such exposure. Among particles present on organic dust, endotoxin is the most related to the inflammation witch cause a numerous respiratory diseases. Endotoxin from gram-negative bacterias are airborne and found in different concentrations in dust and on the ground of home and rural environment, indoor or outdoor living areas. Exposure to endotoxin is unpreventable, but the degree of exposure may vary. Some studies have shown correlation between workers symptoms with degree of exposure. AIMS: Our aim was to evaluate amount of endotoxin exposure in laboratories and animal facilities and to test its association with asthma, rhinitis, and atopy presented by workers. METHODS: This is a cross-sectional study performed in the University of São Paulo, campus of Ribeirão Preto (USP-RP) and in the State University of Campinas (Unicamp). Dust samples were collected from laboratories and animal facilities with rat, mouse, guinea pig, rabbit or hamster. We also sampled workplaces without animals. These samples were analyzed by the method of Limulus amebocyte lysate (LAL). The concentration of endotoxin detected in those workplaces were tested for association with symptoms, bronchial hyperresponsiveness, spirometry data and skin prick test results. RESULTS: One hundred, forty-five workplaces had their dust sampled: 74 (51%) in USP-RP and 71 (49%) in Unicamp. Among those, 92 (63%) workplaces had laboratory animals (57 in USP-RP and 35 in UNICAMP) and 53 (37%) did not have animals. These workplaces had 751 workers or students, 412 were animal handlers and 339 were nonhandlers. Animal handlers workplaces were exposed to higher concentrations of endotoxin 55 ± 79 UE/mg as compared with the non-handlers group 19 ± 27 UE/mg (p < 0.001, Studentst test). We divided endotoxin concentration into two leves: high (> 20.4 UE/mg) and low concentration. The high concentration associated with wheezing prevalence, i.e. 27% of animal handlers exposed to high concentration reported wheezing in the last 12 months (p < 001, chi-squared test). The concentration of endotoxin was not associated with symptoms of rhinitis, atopy or bronchial hyperresponsiveness. CONCLUSION: Exposure to endotoxin has effect on workers respiratory system, although it is not associated with asthma. Higher endotoxin concentration is associated with wheezing. Therefore, animal handlers are prone to present wheezing and preventive measures are necessary for these workers.

Orientador: Sonia Maria Oliani / Coorientador: Cristiane Damas Gil / Banca: Janetti Nogueira de Francischi / Banca: José Roberto Mineo / Banca: Dorotéia Rossi Silva Souza / Banca: Ana Elizabete Silva / Resumo: A proteína anexina A1 (AnxA1) apresenta importantes propriedades anti-inflamatórias e estudos sugerem que suas ações podem ser mediadas por receptores para peptídeos formilados (FPR). Embora os efeitos anti-inflamatórios da AnxA1 e de seus peptídeos miméticos, especialmente o Ac2-26, tenham sido explorados em diversas investigações, são raros os estudos da AnxA1 nas inflamações oculares. Em razão dos graves efeitos colaterais dos tratamentos atuais para a uveíte, uma importante causa de cegueira, investigamos, in vivo, os efeitos e o mecanismo de ação da AnxA1 nos tecidos oculares de roedores na uveíte induzida por endotoxina (EIU). Ratos machos (Rattus novergicus) foram anestesiados e inoculados, por via subcutânea, na pata direita com lipopolissacarídeo (LPS) (100µg) para o desenvolvimento da uveíte. Após, foram divididos em grupos experimentais (n=10/grupo): EIU por 24 e 48h; EIU por 24h e tratados farmacologicamente por administrações tópica e sistêmica do peptídeo Ac2-26 (100µg) e EIU 24h tratado sistemicamente com peptídeo e Boc2, antagonista do FPR (50µg/animal). Para confirmar a importância da AnxA1 endógena na resolução da inflamação ocular, camundongos selvagens e deficientes para AnxA1 (AnxA1-/-) foram induzidos à uveíte por 24h sem tratamento. Nesses animais AnxA1-/- a resposta inflamatória foi exacerbada em comparação com os selvagens. Enquanto, nos olhos dos ratos, as análises quantitativas dos leucócitos, dosagens de interleucina (IL)-1β, IL-6, fator de necrose tumoral (TNF)-α, óxido nítrico (NO) e expressão da ciclo-oxigenase (COX)-2 nos tecidos e/ou no humor aquoso indicaram os efeitos anti-inflamatórios do peptídeo. Efeitos que foram revertidos na presença do Boc2. As análises imuno-histoquímicas das proteínas AnxA1, AnxA1 fosforilada em... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Annexin A1 (AnxA1) is a protein that displays anti-inflammatory properties and some studies suggest that its effects may be mediated by formyl peptide receptors (FPR). Although the anti-inflammatory activities of AnxA1 and its mimetic peptides, including Ac2-26, have been explored in several investigations, the role of AnxA1 in ocular inflammatory processes has not yet been elucidated. Given the common side effects of the current therapies used to treat uveitis, an important cause of blindness worldwide, we investigated, in vivo, the AnxA1 effects and mechanism of action in endotoxin-induced uveitis (EIU). Rattus norvegicus were induced to uveitis (lipopolysaccharide - 100 µg) and divided into experimental groups (n=10/group): EIU untreated for 24 and 48h, EIU treated with topical applications (4/4h) or a single intravenous injection of Ac2-26 (100µg) and EIU systemically treated with the peptide and Boc2, the FPR antagonist (50µg/animal). To confirm the importance of endogenous AnxA1 in the resolution of ocular inflammation, wild-type and AnxA1 deficient (AnxA1-/-) mice were also induced to uveitis without treatment for 24h. AnxA1-/- mice showed exacerbated inflammation compared to wild-type animals. As, quantitative analyses of leukocytes, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, nitric oxide (NO) levels and cyclooxigenase (COX)-2 expression in ocular tissues and/or in aqueous humor of rats eyes revealed the anti-inflammatory effects of the peptide, which were abrogated in the Boc2 presence. Immunohistochemical analysis of AnxA1, serine-or-tyrosine-phosphorylated AnxA1 (AnxA-S27-PO4 - AnxA-Y21-PO4) in the ocular tissues showed AnxA1 and AnxA1-S27-PO4 expression in epithelial (cornea, iris and ciliary processes) and nervous cells. These expressions were increased in... (Complete abstract click electronic access below) / Doutor

As endotoxinas são responsáveis por importantes reações sistêmicas e locais, podendo comprometer o sucesso do tratamento endodôntico. O hidróxido de cálcio, mesmo com limitações, mostra-se como opção única efetiva entre as soluções irrigadoras e as medicações intracanal na redução das endotoxinas nos túbulos dentinários. O experimento realizado avaliou a eficiência do laser de Nd:YAG frente à uma endotoxina inoculada no canal radicular pela utilização de duas cinemáticas de irradiação. Após um período de incubação, confirmada a passagem da endotoxina inoculada no canal radicular através da dentina e do cemento radicular, a irradiação foi realizada ou com cinemática oscilatória ou com cinemática helicoidal, para posterior leitura e comparação com os grupos controle. Os parâmetros utilizados na irradiação com o laser de Nd:YAG de comprimento de onda de 1064 nm foram: 100mJ, 15 Hz, 1,5 W, diâmetro do núcleo da fibra 320 ?m, fluência do pulso 124 J/cm2, largura do pulso 120 ?s e intervalo de relaxação térmica 30 s. Os dados foram comparados pelo método ANOVA, complementado pelo teste de Tukey. As concentrações de endotoxina nos espécimes dos grupos irradiados (helicoidal e oscilatório) foram similares. Além disso, elas também foram similares às dos espécimes controle negativo e significantemente menores (p=0,0271) que as do controle positivo, que não sofreram irradiação. A irradiação com laser de Nd: YAG, nas condições deste experimento é efetiva na inativação da endotoxina da dentina radicular. / The endotoxins are responsible for important systemic and local reactions that could compromise the endodontic success. The calcium hydroxide even with limitations is the only effective option among the irrigant solutions and intracanal medications on the endotoxin reduction in the dentinal tubules. This experiment evaluated the Nd:YAG laser efficacy regarding the inoculated endotoxin in the root canal with two cinematic irradiation procedures. The irradiation was accomplished using an oscillatory and helicoidal technique after an incubation period where the passage of the inoculated endotoxin inside the root canal through dentin and cement was confirmed. Following this the analysis and comparison among the groups was performed. The Nd:YAG laser (?=1064 nm) irradiation, used parameters were: 100mJ, 15 Hz, 1,5 W, core fiber diameter of 320 ?m, pulse fluency of 124 J/cm2, pulse width of 120 ?s and relaxation time interval of 30 s. The data were statistically compared by ANOVA complemented by the Tukey\'s test (p<=0,005). The endotoxin concentration at the irradiated samples, regardless the techniques applied were similar. Moreover these concentrations were also similar to those of negative controls and significantly smaller than those of positive controls that were not irratiated (p=0,0271). The Nd:YAG laser irradiation under this experiment conditions was effective on the root canal endotoxin inactivation.

Em países industrializados, as doenças pulmonares são as que mais se destacam quando o assunto é doença ocupacional. Entre os técnicos, cuidadores de animais, médicos e cientistas, as doenças respiratórias e alérgicas a animais de laboratório representam a principal doença ocupacional. Entre os agentes presentes na sujeira orgânica, as endotoxinas são as mais relacionadas às respostas inflamatórias e causadoras de uma série de doenças respiratórias. As endotoxinas, componentes externos das bactérias gram-negativas, são encontradas em várias concentrações em suspensão no ar ou depositadas na poeira do chão, em materiais e equipamentos, no ambiente domiciliar, urbano, rural e em alguns estabelecimentos. É inevitável a exposição à endotoxina, no entanto, o nível de exposição das vias aéreas pode ser muito variado. Alguns estudos mostraram a correlação dos sintomas apresentados pelos trabalhadores de laboratórios ou biotérios com o nível de exposição às endotoxinas. OBJETIVOS: Avaliar a exposição às endotoxinas, presentes na poeira de laboratórios e biotérios e a sua relação com asma, rinite e atopia apresentadas pelos trabalhadores. MÉTODOS: Trata-se de um estudo transversal, realizado na Universidade de São Paulo, campus Ribeirão Preto (USP-RP) e na Universidade Estadual de Campinas (UNICAMP). Foram coletadas amostras de poeira do chão de laboratórios e biotérios que continham rato, camundongo, cobaia, coelho ou hamster; e em laboratórios e salas administrativas que não tinham contato algum com esses animais. As amostras de poeira foram analisadas e a quantidade de endotoxina foi dosada pelo método Limulus amebocyte lysate (LAL). Esta quantidade foi relacionada com variáveis clínicas dos trabalhadores destes locais (sintomas, reatividade brônquica, espirometria e testes alérgicos). RESULTADOS: Foram coletadas amostras de poeira de 145 locais de trabalho. Destes, 74 (51%) da USP-RP e 71 (49%) da UNICAMP. Noventa e dois (63%) locais de trabalho continham animais de laboratório (57 da USP-RP e 35 da UNICAMP) e 53 não os continham (17 da USP-RP e 36 da UNICAMP). Foram utilizados os dados de 751 trabalhadores, 412 formaram o grupo exposto a animais de laboratório e 339 o grupo não exposto. O grupo exposto a animais de laboratório apresentou maior quantidade de endotoxina, 55 ± 79 UE por mg de poeira, quando comparado com o grupo não exposto, 19 ± 27 UE/mg (p < 0,001 pelo teste t de Student). Quando estratificada, a quantidade de endotoxina em elevada e baixa quantidade, a alta concentração (acima de 20,4 UE/mg) de endotoxina se associou ao relato de sibilos nos últimos 12 meses. Ou seja, 27% dos trabalhores expostos a elevadas concentrações relataram sibilos (p < 0,01 pelo teste do qui-quadrado). Porém, a quantidade de endotoxina não se associou com sintomas de rinite, com atopia, com o teste de hiperreatividade brônquica positiva. CONCLUSÃO: A exposição à endotoxina apresenta efeito no sistema respiratório dos trabalhadores mesmo não tendo se associado à asma. A alta concentração de endotoxina se associou com a presença de sibilos, ou seja, os trabalhadores de biotérios e de laboratórios que tem contato direto com animais de laboratório estão mais susceptíveis a apresentarem sibilos ao longo do ano, sendo necessárias medidas de prevenção para estes trabalhadores. / Respiratory diseases are the most common occupational illnesses in industrialized countries and, among them, asthma and allergies are highly prevalent. Respiratory diseases and laboratory animal allergies represent a major occupational illness among technicians, animal caretakers, doctors and scientists whose work requires such exposure. Among particles present on organic dust, endotoxin is the most related to the inflammation witch cause a numerous respiratory diseases. Endotoxin from gram-negative bacterias are airborne and found in different concentrations in dust and on the ground of home and rural environment, indoor or outdoor living areas. Exposure to endotoxin is unpreventable, but the degree of exposure may vary. Some studies have shown correlation between workers symptoms with degree of exposure. AIMS: Our aim was to evaluate amount of endotoxin exposure in laboratories and animal facilities and to test its association with asthma, rhinitis, and atopy presented by workers. METHODS: This is a cross-sectional study performed in the University of São Paulo, campus of Ribeirão Preto (USP-RP) and in the State University of Campinas (Unicamp). Dust samples were collected from laboratories and animal facilities with rat, mouse, guinea pig, rabbit or hamster. We also sampled workplaces without animals. These samples were analyzed by the method of Limulus amebocyte lysate (LAL). The concentration of endotoxin detected in those workplaces were tested for association with symptoms, bronchial hyperresponsiveness, spirometry data and skin prick test results. RESULTS: One hundred, forty-five workplaces had their dust sampled: 74 (51%) in USP-RP and 71 (49%) in Unicamp. Among those, 92 (63%) workplaces had laboratory animals (57 in USP-RP and 35 in UNICAMP) and 53 (37%) did not have animals. These workplaces had 751 workers or students, 412 were animal handlers and 339 were nonhandlers. Animal handlers workplaces were exposed to higher concentrations of endotoxin 55 ± 79 UE/mg as compared with the non-handlers group 19 ± 27 UE/mg (p < 0.001, Studentst test). We divided endotoxin concentration into two leves: high (> 20.4 UE/mg) and low concentration. The high concentration associated with wheezing prevalence, i.e. 27% of animal handlers exposed to high concentration reported wheezing in the last 12 months (p < 001, chi-squared test). The concentration of endotoxin was not associated with symptoms of rhinitis, atopy or bronchial hyperresponsiveness. CONCLUSION: Exposure to endotoxin has effect on workers respiratory system, although it is not associated with asthma. Higher endotoxin concentration is associated with wheezing. Therefore, animal handlers are prone to present wheezing and preventive measures are necessary for these workers.

Endotoxins (E) are a unique and abundant family of glycolipids located in the outer leaflet of the outer membrane of Gram-negative bacteria. Host immune responses to endotoxin depend on ordered endotoxin-host protein interactions, resulting in delivery of an endotoxin monomer to MD-2 which acts as a potent agonist of Toll-Like Receptor (TLR) 4. Activated TLR4 is unique among TLRs in its ability to mobilize two distinct intracellular signaling pathways: the MyD88- and TRIF-dependent pathways. The regulated action of both pathways is likely important for optimal host immune responses to Gram-negative bacterial infection, but how this is achieved is not well understood Recent studies have indicated an essential role for host CD14 in TRIF-dependent signaling by activated TLR4 but the extent to which these observations reflect a general role of CD14 in endotoxin-triggered TRIF signaling or one more narrowly restricted to the specific endotoxins and/or cell types used is uncertain. We have addressed this question by identifying a novel CD14-independent mechanism for efficient delivery of E monomer to MD-2 and TLR4 activation, that is mediated by endotoxin.albumin complexes. We have used these complexes to demonstrate CD14-independent activation of MD-2⋅TLR4 by a wider range of endotoxin species than previously thought possible and activation of both MyD88- and TRIF-dependent pathways. Taken together, the findings in this thesis indicate that the molecular structure and physical presentation of endotoxin as well as CD14-independent properties of the host cell help determine the extent to which CD14 is required for TRIF-dependent signaling by activated TLR4.

Electrostatic Dust Collectors (EDCs) are a passive sampling method that has not yet been fully validated. Our first study investigated the effect of EDC mailing and EDC deployment in front of and away from heated ventilation on endotoxin concentrations. Endotoxin sampling efficiency of heated and unheated EDC cloths was evaluated. EDCs express mailed cross-country yielded no significant changes in endotoxin concentrations when dust-only samples were compared to high quality control (QC) spiked-EDCs (p=0.21) and low QC spiked-EDCs (p=0.16). EDCs were deployed in 20 apartments with one EDC placed in front of the univent heater and another EDC placed on a built-in bookshelf. Endotoxin concentrations were significantly different (p=0.049) indicating that EDC placement impacts endotoxin sampling. Heated and unheated EDCs were deployed for 7 days in farm homes. There was a significant difference between endotoxin concentrations (p=0.027). The electrostatic charge of 12 heated and 12 unheated EDC cloths were significantly different (p=0.009). These studies suggest that heating cloths may diminish their electrostatic charge and endotoxin sampling capabilities. The EDC sampling time needed to achieve detectable and reproducible loading for bioaerosols has not been systematically evaluated. In our second study, EDCs were deployed in 15 Iowa farm homes for 7-, 14-, and 28-day sampling periods to determine if endotoxin and allergens could be quantified and if loading rates were uniform (i.e. doubling from 7 to 14 days and 14 to 28 days and quadrupling from 7 to 28 days). Loadings between left and right paired EDC cloths were not significantly different and were highly correlated for endotoxin, total protein, and cat (Fel d1), dog (Can f1) and mouse (Mus m1) allergens (p<0.001). EDC endotoxin sampling had close agreement between paired samples (Pearson p=0.96, p<0.001). EDC endotoxin loading doubled from 7 to 14-day deployments but the loading rate decreased from 14 to 28 days of sampling with only a 1.38 fold increase. Allergen exposure assessment using EDCs was less satisfactory. Paired EDCs and daily Button aerosol samplers (BS) were used in our third study to concurrently sample endotoxin in 10 farm homes during 7 day periods in summer and winter. Winter sampling included an optical particle counter (OPC) for particulate size and number concentration data. OPC particulate matter (PM) data were divided into PM2.5 and PM10-2.5. Summer sampling yielded geometric mean and geometric standard deviation values of 0.82 EU/m3 (2.7) for inhalable aerosol BS and 737 EU/m2 (1.9) for EDCs. Winter values were 0.52 EU/m3 (3.1) for BS and 538 EU/m2 (3.0) for EDCs. Seven day endotoxin values of EDCs were significantly and highly correlated with the 7-day BS sampling averages (p=0.70; p<0.001). An Analysis of Variance indicated a 2.37-fold increase in EDC endotoxin concentrations for each unit increase of the ratio of PM2.5 to PM10-2.5. A 10-fold increase in BS endotoxin concentrations was associated with a 12.2-fold increase in EDC endotoxin concentrations. Our fourth study established QC protocols use of EDCs in large field studies. QCs were developed for endotoxin, peptidoglycan, and glucan for analysis alongside the Agricultural Lung Health study EDC samples. The coefficient of variation percentage (CV) for each QC was used to determine variability. For each QC, 20 EDC cloths were analyzed to establish an acceptable range (mean ± 3 standard deviations). Two QCs were established for endotoxin analysis. The high QCs were dust-spiked EDCs with a CV of 29.7%. The low QCs were spiked with E. coli standard and had a CV of 15.6%. One QC was established for peptidoglycan analysis using dust-spiked EDC extracts. Two glucan QCs were established using dust-spiked EDCs with a high CV (51.7%) and yeast-spiked EDCs with a CV of 26.0%. Endotoxin and glucan concentrations of AGLH EDC samples were found to be significantly correlated (p=0.71; p<0.0001). In conclusion, EDCs are an effective passive sampling method for endotoxin exposure assessment in farm homes.

Background: Endotoxin is a lipopolysaccharide located on the outer membrane of the cell wall of Gram-negative bacteria that is widespread in the environment. Although domestic endotoxin has been found to be associated with asthma and wheeze, its association with chronic obstructive pulmonary disease (COPD) is unclear. It is also unknown how environmental exposures influence the relationship between endotoxin and asthma and very few studies have investigated the effectiveness of interventions in reducing endotoxin in the homes of people with asthma. Goals: The goals of this dissertation were to examine 1) the association of house dust endotoxin with chronic bronchitis or emphysema, two phenotypes of COPD, 2) the influence on the relationship between endotoxin and asthma outcomes of environmental factors such as exposure to dog and cat in homes, climate regions, as well as co-exposure to ambient air pollution, and 3) the effectiveness of an environmental intervention in reducing home endotoxin and asthma symptoms in rural Iowa children with asthma. Methods: For the first two goals of this dissertation, data from the 2005-2006 National Health and Nutrition Examination Survey (NHANES) was used. Dust sampled from the bedroom floor and bedding of 6,963 children and adult participants was evaluated for endotoxin at the University of Iowa Pulmonary Toxicology facility using a kinetic chromogenic Limulus amebocyte lysate assay. Data on asthma outcomes and chronic bronchitis or emphysema (CBE) was collected using questionnaires. Home exposure to dog and cat was considered by pet ownership and levels of dog (Canis familiaris 1) and cat (Feline domesticus 1) allergens in house dust. Annual average particulate matter ≤2.5 µm (PM2.5), ozone (O3), and nitrogen dioxide (NO2) concentrations at participants’ residential location were estimated using the Community Multiscale Air Quality (CMAQ) and Downscaler (DS) models. In the third goal, data from the Louisa Environmental Intervention Project (LEIP) study which included schoolchildren 5-14 years-old with active asthma from Louisa and Keokuk counties in rural Iowa was analyzed. The households were block-randomized to receive extensive (education + professional cleaning) or educational intervention. Environmental sampling and questionnaire administration were done at baseline and during three follow-up visits. Results: In the NHANES, the median concentration of endotoxin in house dust was 16.2 EU/mg. In adjusted analysis, house dust endotoxin (log10-endotoxin) was associated with increased odds of CBE diagnosis (OR: 1.27, 95% CI: 1.00-1.61) and chronic bronchitis symptoms (OR: 1.78, 95% CI: 1.01-3.12). Sensitization to inhalant allergens modified the relationship between log10-endotoxin and CBE diagnosis (P(interaction)=0.001), with stronger associations observed in sensitized participants (OR: 2.46, 95% CI: 1.72-3.50). The association of endotoxin with asthma outcomes was different with climate regions of the U.S. Endotoxin was associated with higher prevalence of wheeze outcomes in the past 12 months in subarctic/very cold/cold regions (OR: 1.48, 95% CI:1.19-1.85) and in hot-humid regions (OR: 1.66, 95% CI:1.04-2.65). In hot-humid regions, endotoxin was positively associated with current asthma (OR: 1.56, 95% CI:1.11-2.18), but negatively with sensitization to any inhalant allergens (OR: 0.83, 95% CI:0.74-0.92). Exposure to dog and cat allergens enhanced endotoxin association with current asthma (OR: 2.00, 95% CI: 1.04-3.83, P(interaction)=0.012) and wheeze in the past 12 months (OR: 1.88, 95% CI: 1.32-2.66, P(interaction)=0.016). House dust endotoxin co-exposure with PM2.5 (CMAQ) was synergistically associated with emergency room visits for asthma in the past 12 months (OR: 5.01, 95% CI: 2.54-9.87) in general. In children, a synergistic association was found for co-exposure to house dust endotoxin and NO2 with the outcome (OR: 3.45, 95% CI: 1.65-7.18). In LEIP, 104 asthmatic children from 89 homes were included in the study. In the main analysis, extensive compared to the educational intervention was associated with decreased endotoxin load in farm homes (P-value of main effect for intervention <0.0001) and with less frequent nighttime asthma symptoms (Intervention x visit interaction P-value = 0.044). In exploratory analysis, endotoxin load reduction from baseline was associated with less daytime wheeze (OR: 0.59, 95%CI: 0.38-0.91) and daytime cough (OR: 0.62, 95% CI: 0.40-0.97). Conclusions: House dust endotoxin is associated with obstructive pulmonary diseases. The association of endotoxin with asthma outcomes is stronger in cold regions of the U.S. and is enhanced by exposure to pet allergens and co-exposure to ambient air pollutants such as PM2.5 and NO2 in children. The LEIP study demonstrated that extensive cleaning interventions can be effective at reducing endotoxin in the homes children with asthma and can alleviate their symptoms.

Endotoxin (lipopolysaccharide, LPS) a chemical component of cell wall of gram-negative bacteria, is an important mediator in pathogenesis of sepsis and acute respiratory distress syndrome. It causes production and release of a wide array of mediators including cytokines, chemokines, oxygen free radicals and nitric oxide from neutrophils, macrophages, endothelial cells and epithelial cells through the NF-£eB pathway. LPS increases the permeability of microcirculation, and causes the acute formation of numerous endothelial gaps among venular endothelial cells, resulting in extensive plasma leakage in the inflammatory tissue. Urethan is commonly used as an animal anesthetic for nonrecovery laboratory surgery. It is aslo an £\2-adrenoreceptor antagonist, which can suppress the activation of the cardiovascular system and reduce the angiotensin which increases the blood pressure. Urethan or its metabolites protect animals against LPS, in part, by reducing TNF-£\ release. The aims of the present study to investigate the time-course of vascular permeability in microcirculation of rat trachea, bronchus and esophagus after intravenous application of a high dose of LPS (15 mg/kg), and to reveal the role of urethan (1 g/¢V) and dimethylthiourea (DMTU, 0.375 g/¢V) in inhibition of LPS-induced plasma leakage and edema. India ink was used as a tracer dye to mark leaky microvessels after LPS application. Endothelial gaps were made visible for light microscopy by staining the borders of endothelial cells with silver nitrate. Tracheal sections were stained with toluidine blue to show the subendothelial edema formation. A high dose of LPS was administered intravenously to induce serious plasma leakage and edema and a large number of endothelial gaps formed in postcapillary and collecting venules in the rat trachea and esophagus. The peak values of plasma leakage and edema occurred 5 min after LPS (P<0.01). Urethan anesthesia significantly inhibited LPS-induced plasma leakage by 95 ¡Ó 1.7% in various parts of the respiratory tracts and inhibited edema ratio in the trachea by 57%. Urethan was also found to reduce leukocyte infiltration and the number of endothelial gaps by 46.8 ¡Ó 4.6%. DMTU pretreatment significantly inhibited plasma leakage by 88.5 ¡Ó 2.5% in the respiratory tract and inhibited edema ratio in the trachea by 89% at 5 min after LPS. It is concluded that LPS-induced increase in plasma leakage and edema correlated with the formation of endothelial gaps, and association with activation of alpha 2-adrenergic receptors and hydroxyl free radical production.

¡iAbstract¡j Lipopolysaccharide (LPS) is the toxic chemical component of the cell wall in all gram-negative bacteria which can stimulate immune cells, including macrophages and white blood cell, to release cytokines such as interleukin-1£], interleukin-6 and tumor necrosis factor-£\. These pro-inflammatory mediators induce systemic acute inflammation and multiple organs dysfuction syndrome in sepsis. Plasma leakage from microvasculature is a hallmark of inflammation. Previous studies have demonstrated that other inflammatory agents, such as capsaicin, substance P and histamine could cause the acute formation of numerous endothelial gaps in the venules that result in extensive plasma leakage in the inflammatory tissues of the whole respiratory tract and a part of digestive tract in a few minutes. Mammalian intestines have many goblet cells that synthesize mucus and discharge it into the intestinal lumen. The mucus film that covers the surface epithelium facing the lumen of digestive system, is an immune defense that can prevent gastrointestinal epithelium from chemical and physical damage and act as a lubricant. Changes in goblet cell function and number are involved in microbial infection, inflammatory syndromes and immune factors. This study was aimed to investigate: (1) The degree of Plasma leakage and goblet cell hypersecretion in the small intestine of rats after an intravenous injection of a high dose of LPS (15 mg/kg), and (2) The involvement of vagus nerve and cholinergic receptors in plasma leakage and goblet cell secretion. For the study of plasma extravasation in small intestine during endotoxema, India ink was used as the tracer to mark the inflamed leaky microvessels. Rats were perfusion-fixed through the aorta, and endothelial gaps between endothelial cells of blood vessels were made visible with silver staining. The methacrylate sections of the ileum 3 £gm in thickness were stained with Alcian blue and periodic acid-schiff reagent to detect glycoproteins of goblet cells. Our results showed that LPS not only caused an increase in plasma leakage but also triggered degranulation of many goblet cells in villi and crypts. Numerous gaps were found in postcapillary venules and collecting venules, and plasma extravasation was observed in the serosa and tunica muscularis rat small intestine after LPS. Extensive plasma extravasation occurred in earier phases (5-30 min). However, numerous goblet cells started to discharge mucus granules 30 min after LPS treatment. A large amount of extracellular mucus was accumulated between intestinal villi 1 hour after LPS stimulation. Pretreatment with atropine, the muscarinic receptor antagonist, significantly inhibited goblet cell secretion. The inhibitory effect of pretreatment with atropine or bilateral cervical vagotomy on LPS-induced plasma leakage was not consistent. It is concluded that the plasma leakage and goblet cell hypersecretion induced by endotoxin shock was time-dependent and was associated with activation of muscarinic cholinergic receptors.

Verfasser: Gerald Stumpf Titel: Feldstudie zu fieberhaften Erkrankungen des Pferdes unter besonderer Berücksichtigung der Ehrlichiose und freien Endotoxins Institut: Institut für Bakteriologie und Mykologie der Veterinarmedizinischen Fakultät der Universität Leipzig Eingereicht im Dezember 2005 Bibliographische Daten: 92 Seiten, 13 Abbildungen, 15 Tabellen, 134 Quellenangaben Schlüsselworte: Pferd – Fieber – Ehrlichiose – Endotoxin – CRP – Neopterin – PCR Die vorliegende Arbeit beschäftigt sich mit den in der ambulanten Praxis vorkommenden fieberhaften Erkrankungen des Pferdes. Ein Ziel bestand in der Analyse und Verifizierung der klinischen Beobachtungen in der Praxis und der routinemäßigen Laborparameter. Durch die Verwendung zusätzlicher Parameter wurde deren diagnostische Bedeutung am erkrankten Pferd untersucht, um so den diagnostischen Rahmen bei der ätiopathogenetischen Aufklärung des Fiebers unbekannter Genese zu erweitern. Anhand eigener Untersuchungen von Erkrankungen mit Fieber unbekannter Genese im Vergleich zu anderen fieberhaften Krankheiten des Pferdes wurde die Bedeutung des Fiebers unbekannter Genese beim Pferd herausgestellt und das Krankheitsgeschehen charakterisiert. In die Laboruntersuchungen wurden die Entzündungsparameter Neopterin und CRP zusätzlich einbezogen. Mittels Antikörpernachweisen wurde die Beteiligung von Endotoxinen an der Entstehung von Fieber unbekannter Genese untersucht. Außerdem wurde die PCR zum Nachweis von Ehrlichiose bei spontan fieberhaft erkrankten Pferden eingesetzt. Bei 195 (50,9%) von insgesamt 383 Patienten wurde Fieber unbekannter Genese (Fiebertyp 1) registriert, im Jahresdurchschnitt waren es 65 (56 - 72) Pferde (Tab. 2). Dieser Fiebertyp hatte demnach die größte Bedeutung (p0,05). Es tritt in den Herbst und Wintermonaten am häufigsten auf. Rasse- und Altersprädispositionen ist nicht feststellbar. Es handelt sich um ein mit Fieber einhergehendes Krankheitsgeschehen mit geringer Kontagiosität. Der CRP-Wert liegt bei den Patienten mit Fieber unbekannter Genese im Normbereich. Das bedeutet, in Übereinstimmung mit OKIN et al. (2005), AGRAWAL (2005) und DE MAAT (2004), dass es nicht zu den entsprechenden mit Gewebszerfall einhergehenden Entzündungsreaktionen gekommen war. Das Neopterin wurde hier erstmals bei Pferden mit Fieber unbekannter Genese. Dieser Wert war erhöht (x=7,29). Es ist ein unspezifischer Parameter für den negative Korrelation (r=0,33, p0,05) mit Anti-Endotoxin-IgG festgestellt wurde. Die Erhöhung der Immunglobuline der Klasse IgG (Antiendotoxin-IgM: x=1,18 und Anti-Lipid-A-IgM: x=1,5) weist auf eine Beteiligung freier Endotoxine an der Entstehung von Fieber unbekannter Genese beim Pferd hin. Das Vorkommen von Anaplasma phagocytophilum (Ehrlichia equi) wurde erstmals in Deutschland beim Pferd molekulargenetisch nachgewiesen. Obwohl die ätiologische Diagnosestellung in dieser Studie nur begrenzt erweitert werden konnte, ergeben sich nach der hier vorgenommenen Charakterisierung des Fiebers unbekannter Genese beim Pferd für die Zukunft gute Ansatzpunkte für den gezielten Einsatz der PCR mit selektiv ausgewählten Primern, um den diagnostischen Rahmen zu erweitern. / Author: Gerald Stumpf Titel: Fieldstudy of febrile diseases in the horse in particular condition of ehrlichiosis and free endotxin Institute: Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, University of Leipzig Submitted in December 2005 Bibliographic data: 92 pages, 13 figures, 15 tables, 134 references Key words: Horse – Fever – Ehrlichiosis – Endotoxin – CRP – Neopterin - PCR The importance and prevalence of fever of unknown origin in horses was investigated, characterised and compared with other febrile conditions in horses. Inflammatory and stress parameters such as neopterin and CRP were included in the investigated laboratory profile. The level of IgG was investigated by an immunological approach for checking the involvement of endotoxins in the genesis of fever. PCR was used for the detection of ehrlichiosis in horses with spontaneous fever. In total, 383 horses with febrile conditions were examined within a period of 3 years. In 195 horses (50.9%) fever of unknown origin was diagnosed (fever type 1) indicating that this is the most important fever type in horses (p0.05) mainly seen in autumn and winter. In contrast, an age or race predispostion could not be detected. Typically, this fever type is characterised by a low contagiosity. The horses with fever of unknown oigin showed CRP values within the physiological range. This is indicative for the lack of tissue damage and respective inflammatory processes and supports the findings of OKIN et al. (2005), AGRAWAL (2005) und DE MAAT (2004). To the knowledge of the author, this is the first report on investigations of neopterin in horses with fever of unknown origin. Neopterin is an unspecific parameter and showed a negative correlation (r=0.33, p0.05) with anti-endotoxin IgG. The mean neopterin value in horses with fever of unknown oigin was increased (7.29). The observed increase of anti-endotoxin-IgM and anti-Lipid A-IgM indicates the involvement of endotoxins in the genesis of fever of unknown origin in horses. Moreover, this is the first report about the molecular genetic detection of Anaplasma phagocytophilum (Ehrlichia equi) in horses in Germany. In conclusion, the characterisation of fever of unknown origin in this thesis may stipulate further targeted investigations using PCR with selective primers to improve the etiological diagnosis of this disease.