Freiburg i. Br., Univ., Diss., 2007.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 112-132). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-04-20T15:09:26Z No. of bitstreams: 1 Lethicia Souza Tavares.pdf: 1585867 bytes, checksum: 5409eeb189a05f35fe911992f6371542 (MD5) / Made available in DSpace on 2018-04-20T15:09:26Z (GMT). No. of bitstreams: 1 Lethicia Souza Tavares.pdf: 1585867 bytes, checksum: 5409eeb189a05f35fe911992f6371542 (MD5) Previous issue date: 2017-02-24 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Calotropis procera, is a medicinal plant known in Pernambuco as “silk-cotton”. Is laticífer plant belonging to the family Apocynaceae broadly found in the Brazilian Northeast. Current literature data show that proteins obtained from its latex harbour anti-inflammatory action. In the present study, a protein fraction obtained by ion-exchange chromatography named LPPII, which is rich in cysteine proteases, had its immunmodulatory activity evaluated in cultures of peritoneal macrophages infected by Salmonella enterica Sor. Typhimurium. Macrophages were obtained from the peritoneal cavity of Swiss mice using RPMI culture medium containing antibiotics. The cells obtained were adjusted to 1 x 106 cell/mL and incubated at 37° C and 5% CO2, in cell culture plates, and the adherent macrophages used in the assays. The bacterial quantification assays were performed in a preventive manner, where the macrophages were treated with LPPII (1 μg/ mL or 10 μg/ mL) or LPPII+IAA (10 μg / mL) (inactivated with iodoacetamide) followed by infection with S. Typhimurium (1 x 108 CFU / mL), and in a curative manner, where macrophages were first infected with S. typhimurium (1 x 108 CFU / mL) followed by treatment with LPPII (1 μg/ mL or 10 μg/ mL) or LPPII+IAA (10 μg/ ml). The cell viability assay was performed curatively with macrophages infected with S. Typhimurium (1 x 108 CFU / ml). For IL1β, TNF, IL-6, iNOS and TLR-4 cytokine gene expression assays, macrophages were only treated with LPPII (1 μg/ mL or 10 μg/ mL) or LP+IAA (10 μg / mL), or bacterial LPS stimulation, followed by curative or preventative treatments with LPPII (1 μg/ mL or 10 μg/ mL) or LP+IAA (10 μg/ mL). The results show that macrophages infected with a S. Typhimurium C5 and treated with LPPII in a preventative or curative manner, reduced the number of viable bacteria in the intracellular environment. In this case, macrophages treated preventively with LPPII 10 μg/ mL, showed a significant decrease (p <0.05) in the amount of intracellular bacteria when compared to the control, macrophages without LPPII treatment. In the curative form, significant decrease of intracellular S. Typhimurium was observed in LPPII1μg/ mL-treated macrophages compared to control cells, without LPPII treatment. On the other hand, the groups treated with LPPII+IAA had a greater number of intracellular bacteria, suggesting the action of cysteine proteases on the observed antimicrobial effect. Curative treatment with LPPII also increased the viability of infected macrophages relative to the untreated control groups. Thus, groups treated with LPPII1μg/ mL obtained viability 14.74% greater than the control group without treatment, whereas macrophages treated with LPPII10μg/ mL obtained 24.11%. Groups of macrophages stimulated with LPS and then treated with LPPII had a reduction in the gene expression of the inflammatory cytokines TNF, IL1-β and IL-6, in addition to Toll-like receptor 4. Taken together, we found that LPPII obtained from latex of C. procera presents biomolecules with immunomodulatory activity beneficial to the control of S. Typhimurium infections. / Calotropis procera, planta medicinal conhecida popularmente em Pernambuco como algodão-de-seda. É uma planta laticífera que pertence à família Apocynaceae, sendo encontrada com facilidade no nordeste brasileiro. Dados da literatura corrente mostram que proteínas obtidas de seu látex possuem ação anti-inflamatória. Diante disso, neste trabalho, uma fração proteica obtida por cromatografia de troca iônica, chamada LPPII, rica em proteases cisteínicas, foi avaliada quanto à sua atividade imunomodulatória em culturas de macrófagos peritoneais infectadas por Salmonella enterica Sor. Typhimurium. Os macrófagos foram obtidos a partir da lavagem peritoneal de camundongos Swiss com meio de cultura RPMI contendo antibióticos penicilina e estreptomicina. As células do fluido obtido foram ajustadas a 1 x 106 cél/ mL e incubadas em estufa a 37ºC e CO2 5%, em placas de cultura de células, e os macrófagos aderentes utilizados nos ensaios. Os ensaios de quantificação bacteriana se deram de forma preventiva, onde os macrófagos foram tratados com LPPII (1 μg/mL ou 10 μg/mL) ou LPPII+IAA (10 μg/mL) (inativada com iodoacetamida) seguido de infecção com S. Typhimurium (1 x 108 UFC/mL), e de forma curativa, onde primeiro se deu infecção dos macrófagos por S. Typhimurium (1 x 108 UFC/mL) seguido de tratamento com LPPII (1 μg/mL ou 10 μg/mL) ou LPPII+IAA (10 μg/mL). O ensaio de viabilidade celular foi realizado de forma curativa com macrófagos infectados com S. Typhimurium (1 x 108 UFC/mL). Para ensaios de expressão gênica de citocinas IL1- β, TNF, IL-6, iNOS e TLR-4, os macrófagos receberam apenas tratamento com LPPII (1 μg/mL ou 10 μg/mL) ou LP+IAA (10 μg/mL), ou houve estímulo de LPS bacteriano, seguidos de tratamentos de forma curativa ou preventiva com LPPII (1 μg/mL ou 10 μg/mL) ou LP+IAA (10 μg/mL). Os resultados mostram que macrófagos infectados com uma cepa de S. Typhimurium C5 e tratados com LPPII de forma preventiva ou curativa, reduziram o número de bactérias viáveis no ambiente intracelular. Neste caso, macrófagos tratados de forma preventiva com LPPII 10 μg/mL, demonstraram diminuição significativa (p<0,05) na quantidade de bactérias intracelulares quando comparados ao controle, macrófagos sem tratamento com LPPII. Na forma curativa, observou-se diminuição significativa de S. Typhimurium intracelular em macrófagos tratados com LPPII1μg/mL, quando comparados ao controle, células sem tratamento com LPPII. Por outro lado, os grupos tratados com LPPII+IAA tiveram um maior número de bactérias intracelulares, sugerindo a ação das proteases cisteínicas no efeito antimicrobiano observado. O tratamento curativo com LPPII também aumentou a viabilidade dos macrófagos infectados em relação aos grupos controles sem tratamento. Deste modo, grupos tratados com LPPII1μg/mL obtiveram viabilidade 14,74% maior que o grupo controle sem tratamento, enquanto que macrófagos tratados com LPPII10μg/mL obtiveram 24,11%. Grupos de macrófagos estimulados com LPS e, a seguir, tratados com LPPII, tiveram redução na expressão gênica das citocinas inflamatórias TNF, IL1-β e IL-6, além de receptor do tipo Toll-4. Tomados esses resultados em conjunto, observamos que LPPII obtida do látex de C. procera apresenta biomoléculas com atividade imunomodulatória benéfica ao controle de infecções por S. Typhimurium.

Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T15:02:44Z (GMT). No. of bitstreams: 1 Mendes_GuilhermeMartinesTeixeira_M.pdf: 1976901 bytes, checksum: 9e7c5547a60ab82c824934c40b10ce27 (MD5) Previous issue date: 2008 / Resumo: O gênero Salmonella spp é formado por bacilos gram-negativos, que podem ser divididos em 3 espécies: S. enterica, S. bongori e S. subterranea. A maioria das sorovariedades patogênicas para o homem está incluída no subgrupo I da espécie S. enterica. A infecção por S. enterica inicia-se com a ingestão de água ou alimentos contaminados. Estes microrganismos são patógenos intracelulares facultativos e, uma vez ingeridos, apresentam a capacidade de aderir e invadir células da mucosa intestinal, preferencialmente células M. Uma vez ultrapassada a mucosa intestinal, S. enterica invade, persiste e prolifera no interior de vacúolos de células do sistema retículo endotelial podendo assim, alcançar diferentes órgãos e tecidos do hospedeiro, causando infecção sistêmica. Sendo assim, linhagens mutantes avirulentas, mas ainda capazes de causar infecção transitória, são boas candidatas a potenciais vacinas vivas orais. Tais mutantes são potenciais carreadores de proteínas heterólogas, compondo, assim, as chamadas vacinas multi-valentes. Que seja de nosso conhecimento, não existem mutantes atenuados de S. enterica desenvolvidos inteiramente no Brasil, havendo necessidade de pagamento de patentes a grupos estrangeiros para sua utilização. Em eucariotos, o DNA cromossômico bacteriano está associado a proteínas (histonas) formando o núcleo, enquanto em procariotos, estas proteínas são denominadas histona-like, formando um nucleóide. Dentre essas proteínas podemos citar a IHF (integration host factor), um heterodímero que controla ou influencia vários processos celulares, como a duplicação e recombinação do DNA, além de regular positiva ou negativamente a expressão de vários genes. Neste estudo, mutantes nulos para os genes himA e himD de IHF foram criados pela técnica de recombinação homóloga mediada pelo sistema ? Red (Datsenko e Wanner, 2000) e testados quanto a atenuação da virulência e capacidade de desencadear resposta imune efetiva e protetora contra a salmonelose murina. Os mutantes também foram caracterizados quanto a diversas características biológicas, como a capacidade de invasão e sobrevivência intracelular, resistência a radicais reativos de oxigênio e nitrogênio, ente outras, sendo os resultados comparados com as respectivas linhagens selvagens. Os mutantes himA e himD de S. enterica foram atenuados e capazes de induzir resposta imune protetora quando desafiados com doses elevadas da linhagem selvagem, indicando que estas linhagens recombinantes são potenciais candidatas a vacinais vivas orais / Abstract: The genus Salmonella sp is formed by gram-negative bacilli, which can be divided into 3 species: S. enterica, S. bongori and S. subterranea. The majority of the serovars pathogenic to humans is included in the subgroup I of the S. enterica species. The infection with S. enterica starts either the ingestion of contaminated water or food. These microorganisms are facultative intracellular pathogens and, once ingested, they have the capacity to adhere and invade cells of the intestinal mucosa, with preference for M cells. Then, S. enterica can invade and proliferate within vacuoles of immune cells, particularly macrophages, achieving different organs and tissues of the host, causing systemic infection. Mutant strains of S. enterica with attenuation of the virulence but that are still able to cause a transient infection, are good candidates for potential live oral vaccines. These mutants are also good carriers of heterologous antigens to cells of the immunological system, been able to induce an effective immunological response. To the best of our knowledge, no mutants of this type were developed in Brazil leading to the needed to pay royalties to foreign groups for their use. In prokaryotes the genomic DNA are associated with a number of proteins, the so called histone-like proteins, with structural and regulatory properties, forming the nucleoid. The IHF (Integration Host Factor) is one of the more abundant histone-like in prokaryotes. IHF is a heterodimeric DNA-binding protein that controls a number of cellular processes, such as DNA duplication and DNA recombination and also modulates the expression of different genes. In this work we constructed recombinant strains of S. enterica mutants for the himA and himD genes that encode for the IHF subunits using the ? Red system (Datsenko and Wanner, 2000) and tested for attenuation and immunogenicity. The mutant strains were also characterized and compared to the parental strains for other biological characteristics such as the capacity to invade and proliferate into eukaryotic cells and to survive to different stress conditions. The S. enterica himA and himD mutant strains were attenuated for virulence and able to induce a protective immunity against the wild type strain of S. enterica indicating that these recombinant strains are candidates to formulate a new live oral vaccine / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

No description available.

The aim of this study was to establish a bacteria-induced mouse model of human IBD applicable for investigation of the role of commensal versus pathogenic bacteria in the onset and development of chronic intestinal inflammation. The mouse model introduced in the present work focused on chronic inflammatory reaction triggered by S. enterica infection. Furthermore, differences between the acute and chronic phase of infection were analyzed. Additionally, it was investigated, whether antibiotic treatment prior to infection has a significant impact on the course of the systemic or local mucosal immune response against S. enterica. Due to the significant increase of the IL-22 level reported from IBD patients, and the assumed crucial role of this immunoregulatory cytokine for human IBD a special focus of this study was to analyze the role of IL-22 in the established mouse model of bacteria-induced chronic colitis.

Food borne infections due to Salmonella enterica serovar Enteritidis (S. Enteritidis) has shown a dramatic increase in many countries. Different egg pasteurisation treaments have been developed in the past but are still not providing practical or optimal solutions. A method is required that ensure that eggs are microbiologically safe, that does not affect the functional quality and possibly also extend the egg shelf life. This research project formed part of a larger study, “Project 32438: The development of a novel microwave system for the pasteurisation of raw whole shell eggs” funded by the National Research Foundation Innovation Fund and conducted by a consortium consisting of the Council for Scientific and Industrial Research (CSIR), Delphius Technologies, Eggbert Eggs and University of Pretoria. One of the phases in the development of the microwave system was an evaluation of the effectiveness of applying different microwave power levels (250W and 300W) on eliminating or reducing S. Enteritidis as well as evaluating the effect on the functional properties of the eggs. The microbiological tests were conducted by the CSIR while the latter evaluation was the focus of the study reported here. Microwave pasteurised eggs had lower foaming capacity but higher Haugh values than control (unpasteurised) eggs. Albumin foam stability did not differ between control and microwave pasteurised eggs and the pH of the albumin was almost similar. The yolk pH of pasteurised eggs was higher than that of unpasteurised eggs. Significant differences were found for the sensory properties of broken out eggs as evaluated by a trained sensory panel. At 300W, pasteurised eggs collected from the left side of the oven had partially coagulated albumin that was not clear. The visual appearance of pasteurised eggs at 300W from left side was more adversely affected than the eggs collected from the right side oven position and all eggs pasteurised at 250W. The albumin foaming capacity, visual appearance and sensory properties of raw eggs pasteurised at 250W were slightly affected by microwave heating. A triangle taste test showed that there was a significant difference between control and pasteurised (300W) eggs. A home use consumer test showed that control and microwave pasteurised (250W) eggs were equally acceptable. Pasteurisation could extend the shelf life of whole shell eggs (WSE) by reducing or destroying spoilage microorganisms. Another phase of the project therefore focused on obtaining background data pertaining to the shelf life of eggs. The effect of coating of egg shells with mineral oil on the functional properties and shelf life of WSE stored at 16ºC (58 % RH); 25ºC during the day and 15ºC at night (55% RH) and 32ºC (32 % RH) for a period of six weeks, were evaluated. These conditions were selected to reflect typical temperatures that are used for storing eggs in South Africa. Haugh units of eggs decreased with storage time at all storage conditions but for coated eggs it decreased at a slower rate. The pH of both the yolk and albumin of coated shell eggs was lower than that of uncoated shell eggs. Coating did not have an influence on the foam stability of egg albumen. Foaming capacity of albumen was negatively affected by oil coating. Coated shell eggs stored at the three conditions had a prolonged shelf life compared to uncoated eggs stored in the same manner. If the prototype microwave oven can be optimised to ensure even distribution of microwaves, microwave pasteurisation of shell eggs has potential to become a significant break through in the poultry industry. / Dissertation (MInstAgrar)--University of Pretoria, 2007. / Food Science / unrestricted

Contaminated poultry and egg products are major sources of Salmonella enterica serovar Enteritidis (S. enteritidis, SE) infections in humans. Colonization of SE in chicken reproductive tract results in the production of contaminated commercial shell-eggs and fertilized hatchery eggs. The complex pathogen-host interactions during SE colonization of chicken reproductive tract are largely unknown. This study was aimed at determining the pathogenic roles of the type three secretion systems (TTSS-1 and TTSS-2) in SE infection of chicken oviduct epithelial cells (COEC). A series of SE strains carrying mutations in the genes encoding structure or effector proteins of TTSS-1 and TTSS-2 were constructed. The invasiveness and intracellular survival rate of each SE strain as well as the host innate immune responses induced by the infections were evaluated. The results demonstrate that both TTSS-1 and TTSS-2 are required by SE to invade COEC which involve genes encoding effector proteins SipA, SopB, SopE2, and PipB. In addition to their involvement in host cell invasion, sipA and sipB are also necessary for the survival or replication of SE inside COEC. Inactivation of TTSS-2 genes (ssaV and pipB) resulted in an enhanced bacterial proliferation inside COEC. The data from this study also show that SE infection triggers pro-inflammatory responses in COEC and TTSS-1 is involved in the expression of iNOS and IL-8, a CXC chemokine. TTSS-1 and TTSS-2 are not necessary for induction of K203, MIP-1β, and IL-10 or suppression of TGF-β3 in COEC.

Opioids have been shown to modulate immune function in a variety of assays and animal models. In a more limited number of studies, opioids have been shown to sensitize to infection. Heroin, the prototypical opioid drug of abuse, is rapidly metabolized to morphine in the body. Morphine has been used as an analgesic for hundreds of years, and continues to be a drug of choice for treating pain in ICU and trauma patients. The continued use of these opioid compounds in humans warrants further investigation of their effect on immune responses against, and progression of, common bacterial infections. Two infections were investigated in this thesis using murine models, Acinetobacter baumannii and Salmonella typhimurium. A recent increase in the prevalence of A. baumannii infections among healthy, but wounded, military personnel, lead to the hypothesis that analgesic morphine might sensitize to infection with this multiply-drug resistant bacterium. A systemic, intraperitoneal A. baumannii infection model was established in mice that resulted in rapid, disseminated disease where animals became septic as organisms replicated in the blood, lungs, and other organs. This model was used to investigate the role of various parameters of innate immune defenses to Acinetobacter. Neutralization of neutrophils by antibody depletion greatly sensitized to this infection. Infection resulted in a rapid, biphasic induction of both IL-17 and the chemokine, KC/CXCL1, a major chemotactic factor for neutrophils, that continued to rise through 18h after bacterial inoculation. However, depletion of either IL-17 or KC/CXCL1 using monoclonal antibodies failed to sensitize to Acinetobacter infection. Further, IL-17 receptor KO mice were not sensitized to this infection. Collectively, these results suggest that there must be other chemotactic factors for neutrophils that can compensate for the absence of IL-17 and KC. Morphine, delivered by extended release pellet, sensitized two strains of mice to two strains of Acinetobacter, as measured by mortality to a sublethal challenge dose, and this effect was blocked by administration of the opioid-receptor antagonist, naltrexone. . Morphine increased Acinetobacter burdens in the organs and blood of infected mice, and increased the levels of pro-inflammatory cytokines. Evidence for an effect of morphine on neutrophil infiltration was obtained. Morphine decreased the total numbers of cells, as well as the total numbers of neutrophils and macrophages infiltrating into the peritoneal cavity. This inhibition of neutrophil accumulation correlated with suppression of levels of both IL-17 and KC/CXCL1. The evidence supports the conclusion that morphine sensitizes to Acinetobacter infection by suppressing the response of neutrophils, potentially via depression of neutrophil chemotactic factors IL-17 and KC. However, taken together with the data above there are probably additional factors in addition to IL-17 and KC that are sensitizing the animals to infection in the presence of morphine. In addition to these studies, the opioid-receptor dependency of morphine-mediated sensitization to Salmonella enteric serovar Typhimurium was examined. Previous experiments had determined that extended release morphine pellets sensitized mice to a sublethal dose of Salmonella, as determined by survival and bacterial burdens in the organs of infected mice, but naltrexone resulted in only incomplete reversal of the morphine-mediated effects. To further characterize the receptor dependency of the observed phenomenon, mu-opioid receptor knockout (MORKO) mice were used. MORKO mice were found to be completely resistant to the lethal effects of morphine plus infection observed in wild-type (WT) mice. In addition, MORKO mice showed greatly reduced bacterial burdens and pro-inflammatory cytokine levels when treated with morphine and challenged with a sublethal challenge dose of Salmonella, in comparison to WT mice. In summary, the studies presented in this thesis explored basic mechanisms of innate immunity to A. baumannii using a systemic model of infection. The work provides additional evidence that morphine sensitizes to infection, using models of Acinetobacter and Salmonella in mice. An implication of this work is use of caution in the administration of opioids in patients that are susceptible to opportunistic infections. / Microbiology and Immunology

Cinnamic acid (CA) is a naturally occurring organic acid that is found in some fruits and a number of spices. CA has antimicrobial activity against certain spoilage microorganisms and pathogenic bacteria. However, the acid is poorly soluble in water. Cyclodextrin molecules have a hydrophobic cavity that allows them to serve as a host for insoluble molecules in aqueous matrices. This study was conducted to determine if the aqueous solubility of cinnamic acid could be improved via complexation with α- or β-cyclodextrins, and if these complexes could be used to control bacterial pathogens in juices. Based upon phase solubility analysis, α-cyclodextrin was chosen as the host molecule for the remainder of this study. In complex with α-cyclodextrin, the solubility of cinnamic acid increased from approximately 400 mg/L to 3800 mg/L. Prepared cinnamic acid complexed with α-cyclodextrin was aseptically added (400 mg/L and 1000 mg/L) to orange juice inoculated with a Salmonella enterica (7 log CFU/mL) and apple cider inoculated with Escherichia coli O157:H7 (7 log CFU/mL). Cider and orange juice samples were extracted on day 0 and at 24 h intervals for seven days and spread plated onto Tryptic Soy Agar. Cinnamic acid was effective for reducing populations of both bacterial pathogens in juice. Populations of E. coli O157:H7 in the apple cider were significantly reduced after 7 days at 25.6 ± 0.42°C at concentrations of 400 mg/L (5-log CFU/mL reduction) and 1000 mg/L (6-log CFU/mL reduction) cyclodextrin-cinnamic acid. S. enterica counts were also reduced in orange juice at 4° C treated with 400 mg/L (2.7-log CFU/mL reduction) and 1000 mg/L (3.2-log CFU/mL reduction) complexed cinnamic acid. The much improved solubility of this compound provides food processors with greater flexibility in using cinnamic acid in their product formulations. / Master of Science