Made available in DSpace on 2014-08-20T13:42:05Z (GMT). No. of bitstreams: 1 Dissertacao_Milena_Tomasi_ Bassani.pdf: 628517 bytes, checksum: af4a756ae84e13ef34c451b2d486162e (MD5) Previous issue date: 2009-10-27 / he accessory gene regulator (agr) is a S. aureus global regulator of virulence factors, as the staphylococcal enterotoxins (SE), responsible for the staphylococcal food poisoning. There are four different agr groups due to the polymorphism in the amino acids sequence of the agrC and agrD. In the literature is described a relationship among agr groups and virulence factors, diseases, preferential host and antibiotic resistance. In this context, it was aimed to characterize the agr groups through biplex PCR, and relationship among agr groups, enterotoxigenic and antimicrobian profiles of S. aureus isolated from foods and bovine mastitis milk. A total of 115 strains were used to characterize the agr groups , being 30 isolated from f bovine mastitis milk and 85 isolated from several sources of foods. To assess the relationship between agr groups and enterotoxin production were used 14/85 strains previously characterized for the presence of some enterotoxins (sea, seb, sec, sed e cluster egc). To determine the profile of antibiotics resistance were used 71/115 strains. We observed a prevalence of agr group II with19.1% (22/115 strains), followed by the agr I with 8.6% (10/115 strains), agr III with 7.8% (9 / 115 strains), and agr IV with6.0% (7 / 115 strains). Among the strains isolated from bovine mastitis milk agr group I was prevailed with 20% (6/30 strains), whereas in the strains isolated from several food sources was observed prevalence of agr group II with 32.7% (18/85 strains), especially among those from chicken meat. Among the 14 strains (14/85) that contained enterotoxin genes, the majority of them contained the cluster egc (70%) belonged to agr II, whereas no relationship was found with those who had the genes for the classical SE (sea, seb, sec, sed). Considering the antibiotic resistance 100% of bovine mastitis milk strains and from various sources of food were resistant to penicillin, ampicillin, cefoxitin, and vancomycin. Relationship was observed between food strains, which were resistant to vancomycin and agr II, however, no relationship was found between antibiotic profile and agr groups among the strains isolated from bovine mastitis milk. These results demonstrated the prevalence of agr II among food strains and agr I among bovine mastitis milk strains. Moreover, the strains that carried the cluster egc were predominant agr II, which could indicate the occurrence of a clonal group among those. Another important result obtained in this study was the high rate of S. aureus multiresistant strains isolated from food, which emphasizes the importance of dissemination of these strains among foods. / O accessory gene regulator (agr) é um regulador global de fatores de virulência em S. aureus, como as enterotoxinas estafilocócicas (EE), responsáveis pela intoxicação alimentar estafilocócica. São conhecidos quatro distintos grupos agr devido ao polimorfismo na seqüência dos aminoácidos de agrC e agrD. Na literatura descreve-se relação entre fatores de virulência, patogenias, hospedeiro preferencial e perfil de resistência a antibióticos com grupos agr. Neste contexto, objetivou-se caracterizar grupos agr através de biplex PCR, e relacioná-los com os perfis enterotoxigênico e antimicrobiano de cepas de S. aureus isoladas em alimentos e leite de vacas mastíticas. Para caracterização dos grupos agr foram utilizadas 115 cepas, sendo 30 isoladas em leite de vacas mastíticas e 85 em diversas fontes de alimentos. Para a relação entre grupos agr e produção de enterotoxina foram utilizadas 14/85 cepas previamente caracterizadas quanto à presença de alguma enterotoxina (eea, eeb, eec, eed e cluster egc), já para determinar o perfil de resistência a antibióticos utilizaram-se 71/115 cepas. Observou-se uma prevalência do grupo agr II, com 19,1% (22/115 cepas), seguido do agr I, com 8,6% (10/115 cepas), agr III 7,8% (9/115 cepas), e agr IV, 6,0% (7/115 cepas). Entre as cepas isoladas em leite de vacas com mastite houve predomínio do grupo agr I, com 20% (6/30 cepas); já nas cepas isoladas de diversas fontes de alimentos observou-se prevalência do grupo agr II, com 32,7% (18/85 cepas), especialmente entre as provenientes de carne de frango. Entre as 14/85 cepas que carreavam genes de enterotoxinas, a maioria que albergava o cluster egc (70%), pertencia ao grupo agr II, enquanto nenhuma relação foi observada com aquelas que possuíam os genes para as EE clássicas (eea, eeb, eec, eed). Com relação ao perfil de resistência antimicrobiana, 100% das cepas isoladas de leite de vacas com mastite e das diversas fontes de alimentos apresentaram resistência à penicilina, ampicilina, cefoxitina e vancomicina. Observou-se relação entre cepas isoladas de alimentos, que eram resistentes à vancomicina e grupo agr II, entretanto, nenhuma relação foi observada entre perfil antimicrobiano e grupos agr entre as cepas isoladas em leite de vacas com mastite. Através destes resultados demonstra-se a prevalência do grupo agr II entre as cepas isoladas de alimentos e do grupo agr I em cepas isoladas de leite de vacas mastíticas. Além disso, nas cepas que carreiam o cluster egc houve predominância do grupo agr II, podendo indicar um grupo clonal entre essas. Outro resultado relevante obtido neste estudo foi à elevada taxa de cepas de S. aureus multiresistentes isoladas em alimentos, o que ressalta a importância da disseminação de cepas multiresistentes entre os alimentos.

As condições de produção de queijos frescos são favoráveis à ocorrência e à produção de enterotoxinas produzidas por estafilococos, sendo estes os principais micro-organismos relacionados aos casos de intoxicação alimentar no mundo, tornando-se necessários estudos sobre a rastreabilidade das fontes de contaminação durante a fabricação, identificação genotípica e habilidade de cepas em produzir enterotoxinas. Este trabalho teve como objetivo isolar e identificar estafilococos positivos para o teste de coagulase, potencialmente produtores de enterotoxinas em laticínios do estado de São Paulo, desde o leite recebido até o produto final. A técnica da mPCR foi utilizada para identificar três espécies de Staphylococcus coagulase-positiva (S. aureus, S. hyicus e S. intermedius) entre os isolados obtidos de diferentes pontos de processamento em laticínios do Estado de São Paulo. O perfil genético das cepas foi avaliado através da comparação de bandas pela técnica Enterobacteria Repetitive Intergenic Consensus (ERIC-PCR) e graficamente demonstrados por um dendrograma. O DNA de 102 cepas isoladas, de amostras de leite cru, leite pasteurizado, coágulo, manipulador, superfícies de equipamentos de laticínios do Estado de São Paulo e queijos foi extraído e submetido à reação em cadeia da polimerase (PCR), utilizando-se primers específicos para a detecção de fragmentos dos genes envolvidos na síntese das toxinas (SE) do tipo A, B, C, D, E, G, H, I e J. Das 102 cepas avaliadas, 5 apresentaram amplificação do fragmento do gene responsável pela codificação da toxina A, 3 para toxina C, 56 para toxina G, 59 para toxina H, 9 para toxina I, e nenhuma para toxinas B, D, E e J. Amostras de leite cru, leite pasteurizado e queijos produzidos nos laticínios e dos isolados de Staphylococcus spp. coagulase positiva que apresentaram ao menos algum dos genes relacionados à produção de toxinas clássicas (A, B, C, D e E) foram submetidos a um teste de detecção de enterotoxinas pelo sistema VIDAS® Staph Enterotoxin (SET2). Os dados obtidos para a identificação molecular das cepas, da ocorrência de cepas portadoras dos genes relacionados à produção de enterotoxinas e da produção fenotípica das enterotoxinas foram submetidos ao teste qui-quadrado. O presente trabalhou confirmou que o risco de intoxicação estafilocócica é real, pois foi encontrada enterotoxina em amostras de leite pasteurizado e queijos. / The production conditions of fresh cheese are favorable to the occurrence and production of enterotoxin produced by Staphylococci, which are the main microorganisms related to food poisoning cases in the world, requiring studies to trace the sources of contamination during manufacturing and genotypic identification ability of strains to produce enterotoxin. This study aimed to isolate and identify staphylococci positive for coagulase test, potentially producing enterotoxins in dairy products in the state of São Paulo, from milk receiving to final product. In three dairy producers of Minas fresh cheese, samples were collected from various points in the manufacturing process. the technique of mPCR was used to identify three coagulase-positive species (S. aureus, S. hyicus and S. intermedius) between isolates from different points of a processing dairy in the state of São Paulo. The genetic profile of the strains were evaluated by comparing the bands through the technique Enterobacteria Repetitive Intergenic Consensus (ERIC-PCR) and were graphically displayed by a dendrogram. The DNA of 102 strains isolated from samples of raw milk , pasteurized milk , curd, handler and equipment surface from dairies in São Paulo State and cheese were subjected to the polymerase chain reaction (PCR), using primers for the detection of specific fragments of the genes involved in the synthesis of toxins (SE) of type A, B , C , D, E, G , H, I and J. Of the 102 strains tested, five showed amplification of the gene fragment encoding toxin A, three for toxin C , 56 to toxin G, 59 to toxin H, 9 to toxin I, and none for toxins B D, E and J. For confirmation, each isolated strain carrying at least some of the genes related to the production of classical enterotoxinas, cheese and milk samples was subjected to detection by enterotoxigenic VIDAS® Staph Enterotoxin II (VIDAS SET2) bioMérieux. This identification reinforces the need to adopt proper hygiene practices in the dairy, avoiding thus the spread of these microorganisms and the possible production of enterotoxin . The data obtained for the molecular identification of the strains, the occurrence of strains carrying the genes related to the production of enterotoxin production and phenotypic enterotoxins were subjected to the Chi-squared test. This work confirmed that the risk of staphylococcal food poisoning is real, because enterotoxin was found in samples of pasteurized milk and cheeses.

Silicon-based electric chip arrays were developed for characterization of Bacillus cereus with respect to the capacity to produce toxins involved in food poisoning and foodborne infections. Bacteria of the B. cereus group contain different sets of four toxins encoded by eight genes. The purpose of this work was to develop a fast method for determination of the presence of these genes in colonies from primary enrichment cultures. The specific DNA detection was based on immobilization of DNA capture probes, which hybridize to specific sites on the target genes. Biotin-labeled detection probes were designed to hybridize with the target DNA adjacent to the capture probes. An extravidin - alkaline phosphatase complex was subsequently bound to the hybridized detection probes. Finally, p-aminophenyl phosphate was added as substrate for the enzyme, and the product p-aminophenol was brought in contact with the interdigitated gold electrode on the silicon chips surface. The p-aminophenol was oxidized at the anode to quinoneimine, which was then reduced back to paminophenol at the cathode. This redox recycling generates a current that was used as the DNA-chip response to the target DNA. Two versions of the assay were used. In the first version the capture probes were immobilized on magnetic beads and all chemical reactions until and including the enzymatic reaction took place in an eppendorf tube while the redox recycling was used to measure the amount of paminophenol produced after transfer from the tube to the silicon chip surface. In the second version a silicon chip array was used with 16 parallel electrode positions, each activated by immobilization of one type of capture probes on the gold electrodes. With this system all chemical reactions took place at the chip surface. The kinetics of cell disruption and DNA fragmentation from B. cereus by ultrasonication was determined. Maximum cell disruption was achieved within 5 min and the chip response increased in proportion to the ultrasonic time. Further ultrasonication up to 10 min resulted in further increasing current although no further cell disruption was observed. If the sonication time was extended above 10 min the signal declined. Based on analysis of the DNA size distribution by early end-point PCR and gel electrophoresis, it is suggested that the first 5 min ultrasonication increased the signal by increasing the release of target DNA molecules. Thereafter the signal was increased by fragmentation of target DNA which increases the diffusion rate and also the accessibility of the hybridization site. Finally, the DNA fragment sizes approached that of the hybridization site (51-bp) which may reduce the signal because of cleavage of the target DNA in the hybridization region. These studies were performed with the bead-based hybridization assay. The assay was highly specific to the target gene (hblC) of both B. cereus and B. thuringiensis with no response from negative control cells of B. subtilis. The 16 positions of the silicon chip array were activated by immobilization of all known toxin-coding genes of B. cereus and also included both a positive control and a negative control electrode positions. When these chips were exposed to ultrasonicated B. cereus, the gold electrodes were fouled by some component in DNA cell lysates. To circumvent this, the released large DNA was first extracted and then ultrasonicated again, since the extract mainly contains large molecular weight DNA. This DNA extract was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the DNA chip arrays. The results agreed with PCR control analysis which means that these electric DNA chip arrays can be used to characterize bacterial colonies with respect to the genes coding of all known toxins of B. cereus: haemolysin (hblA, hblC, hblD), non-haemolytic enterotoxin (nheA, nheB, nheC), cytotoxin K-2 (cytK-2), and cereulide (ces). The chip assay required about 30 min after application of DNA samples. Due to the generic properties of the chips, this technique should also be applicable for characterization of the pathogenicity potential of many other organisms. Keywords: Bacillus cereus, haemolysin, non-haemolytic enterotoxin, cytotoxin K-2, cereulide, toxin-coding genes, bacterial colony, electric DNA chip, ultrasonication, DNA fragmentation. / QC 20101111

Bakterielle Toxine stellen eine wirkungsvolle und effektive Alternative zur Therapie von Tumorerkrankungen dar. Das vom Clostridium perfringens Typ A produzierte Clostridium perfringens enterotoxin (CPE) gehört zu der Gruppe der porenbildenden Toxine und weist eine rezeptorspezifische zytotoxische Wirkung auf, welche über die Membranrezeptoren Cldn3 und Cldn4 entfaltet wird. Diese liegen vor allem in Epithelialkarzinomen wie dem Brust-, Prostata-, oder Kolon-, sowie dem Pankreaskarzinom (PK) stark hochreguliert vor. Ziel dieser Arbeit war die Anwendung des neuen selektiven und effizienten „Onkoleaking“ Suizid-Gentherapie Konzepts für die Behandlung von Cldn3 / 4 überexprimierender PK unter Verwendung eines nicht-viralen translations-optimierten CPE exprimierenden Vektors (optCPE). Weiterhin sollte in dieser Arbeit der genaue molekulare Mechanismus der CPE-vermittelten Zytotoxizität in vitro und auch in vivo analysiert werden. Für die in vitro Analysen wurden verschiedene humane PK Zelllinien, Patienten abgeleitete Xenotransplantate (PDX) und deren abgeleiteten Zellen bezüglich ihrer Cldn3 / 4 Expression und Sensitivität sowohl gegenüber rekombinantem CPE (rekCPE) als auch nach optCPE Gentransfer untersucht. Es konnte eine positive Korrelation zwischen der Effizienz CPE vermittelter Zytotoxizität und der Höhe der Cldn3 / 4 Überexpression gezeigt werden. Des Weiteren wurde die Verfügbarkeit und Zugänglichkeit der CPE Rezeptoren für die Toxinbindung als kritischer Faktor für die durch Porenbildung induzierte Zytotoxizität beschrieben. Auch eine detaillierte Analyse verschiedener apoptotischer und nekrotischer Signalwege und deren Schlüsselmoleküle waren vom besonderen Interesse. Von noch größerer Wichtigkeit war jedoch die Anwendbarkeit und der Nachweis der antitumoralen Wirksamkeit der optCPE-basierten Suizid-Gentherapie mit Hilfe des intratumoralen Jet-Injektion Gentransfers in verschiedenen Luziferase-exprimierenden CDX und PDX Modellen des PK. Alle in vivo Studien zeigten eine selektive optCPE vermittelte Verminderung der Tumorvitalität in Verbindung mit Nekrose, die in fast allen Fällen mit einer Reduktion des Tumorvolumens einher ging. Die tierexperimentellen Studien belegen damit die Effektivität der CPE-basierten Gentherapie im Pankreaskarzinom. Mit diesen neu gewonnenen Erkenntnissen zum „Onkoleaking“ Konzept der CPE Suizid-Gentherapie und deren Wirkungsmechanismen sind Kombinationen mit konventionellen Therapien möglich. / Bacterial toxins have evolved to an effective therapeutic option for cancer therapy and numerous studies demonstrated their antitumoral potential. The Clostridium perfringens enterotoxin (CPE), produced by the anaerobic Clostridium perfringes bacteria, is a pore-forming (oncoleaking) toxin, which binds to its receptors claudin-3 and -4 (Cldn3 / 4) and exerts a selective, receptor-dependent cytotoxicity. The transmembrane tight junction proteins Cldn3 and Cldn4 are known CPE receptors and are highly upregulated in several human epithelial cancers such as breast, colon, ovarian and pancreatic cancer. This study aimed at the evaluation of the potential of oncoleaking gene therapy using a non-viral translation optimized CPE vector (optCPE) as a new suicide approach for the treatment of Cldn3  /  4 overexpressing pancreatic cancer (PC) in vitro and in vivo. We demonstrated the successful in vitro use of optCPE gene transfer in a panel of human PC cells and more importantly patient derived PC xenograft (PDX) derived cells. We showed significant reduction of cell viability in all Cldn3 / 4 overexpressing PC cells after optCPE transfection. Furthermore a positive correlation between CPE cytotoxicity and level of claudin expression was shown. We revealed accessibility of CPE receptors for toxin binding as determining for optCPE mediated cytotoxicity. Since investigation of optCPE induced cell death mechanism was of particular interest, detailed analyses of apoptotic and necrotic key players were performed. By this, caspase dependent- and independent apoptosis and necrosis activation after gene transfer was demonstrated, which was dependent on amount of expressed optCPE and accessibility of Cldn. More importantly, this study demonstrated the applicability and antitumoral efficacy of optCPE gene therapy by the non-viral intratumoral jet-injection gene transfer in vivo in different luciferase-expressing CDX and PDX pancreatic cancer models. The animal experiments demonstrated the selective CPE mediated tumor growth inhibition, associated with reduced tumor viability and effective induction of tumor necrosis. This further corroborated the advantages of this novel oncoleaking strategy. With this gain of knowledge about our new oncoleaking concept of suicidal gene therapy and its mechanism of action, novel combinations with conventional therapies are possible to further improve therapeutic efficacy and to overcome resistance in pancreas carcinoma.

Introdução: O tratamento da Rinossinusite Crônica (RSC) tem sofrido poucos avanços nas últimas décadas. Uma das barreiras na aquisição de novas terapias é a falta de conhecimento pleno sobre sua fisiopatogenia. A carência de avanço decorre principalmente da complexa e provável multifatorialidade da RSC, associada à inexistência de um bom modelo animal que possa mimetizar os fenômenos biológicos que ocorrem em humanos. A maioria dos modelos animais de RSC descrita na literatura mimetiza uma infecção aguda ou promove bloqueio das vias de drenagem que, na maioria das vezes, não corresponde aos mecanismos encontrados nas RSC em humanos. Por outro lado, diversas evidências indicam que as bactérias exercem importante papel na fisiopatogenia da RSC, possivelmente pela presença de biofilmes ou indução de inflamação crônica promovida por endo e exotoxinas. Objetivo: Neste estudo avaliou-se a viabilidade de um modelo experimental de RSC em coelhos, utilizando-se a exposição crônica de toxinas bacterianas em animais previamente sensibilizados à ovalbumina (OVA), analisando seus efeitos histopatológicos sobre a mucosa nasossinusal. Material e Métodos: Após indução de sensibilização com injeção subcutânea de OVA 2,5% e 0,4% de hidróxido de alumínio por duas semanas, os coelhos foram submetidos à implantação de cateter de longa duração em seio maxilar direito. Após, foram submetidos à irrigação nasossinusal com OVA 2,5% três vezes por semana, por duas semanas, e em seguida, irrigação de soluções contendo diferentes toxinas bacterianas (enterotoxina estaflocócica B (SEB) 1 ?g/mL, lipopolissacáride (LPS) 100 ng/mL e ácido lipotecóico (LTA) 100 ng/mL) por quatro semanas. Os animais foram sacrificados 24 horas após a última irrigação e a mucosa do seio maxilar direito (teste) e esquerdo (controle interno) foi coletada para avaliação histopatológica. Resultados: A exposição nasossinusal ao SEB causou espessamento epitelial, infiltração celular, eosinofilia e neutrofilia tecidual, além de redução do epitélio ciliado. A exposição ao LPS causou espessamento epitelial e subepitelial, infiltração celular, eosinofilia epitelial e subepitelial e aumento da fibrose subepitelial. O LTA causou espessamento epitelial e subepitelial, infiltração celular e eosinofílica subepitelial e aumento da fibrose subepitelial. Conclusão: A exposição crônica de toxinas bacterianas na mucosa nasossinusal promoveu alterações histológicas, como espessamento da mucosa e infiltração celular, semelhantes às encontradas em pacientes com RSC. O presente estudo demonstrou que este é um modelo animal viável de RSC. Mais estudos serão necessários para elucidar se os mecanismos patogênicos deste modelo são semelhantes aos observados em humanos. / Background: The treatment of chronic rhinosinusitis (CRS) has had little evolvement in the last decades. One of the barriers to the development of new therapies is the lack of knowledge about CRS pathophysiology. The complexity and multifactoriality of this disease, together with the inexistence of a proper animal model of CRS, are probably the causes for the few advances in CRS therapy. Most of the animal models of CRS resemble acute infection or promote sinonasal obstructions, which are not a very common etiologies in CRS patients. However, there has been a lot of evidence that bacteria play an important role in the pathophysiology of CRS, probably due to the presence of biofilms, or the chronic inflammation induced by endo e exotoxins. Objective: This study aims to evaluate the viability of an experimental model of CRS in rabbits through the use of bacterial toxins in previously sensitized animals with ovalbumin, analyzing its histopathological effects onto the sinonasal mucosa. Materials and Methods: After inducing ovalbumin (OVA) sensitization by intradermic injection of OVA 2,5% and 0,4% aluminum hydroxide for 2 weeks, rabbits underwent maxillary sinus instillation of OVA 2,5% three times a week for 2 weeks followed by sinus lavage with either one bacterial toxin (Staphylococcus aureus enterotoxin B (SEB) 1 ?g/mL, lipopolysaccharide (LPS) 100 ng/mL, lipoteichoic acid (LTA), 100 ng/mL) for 4 weeks. Rabbits were euthanised 24 hours after the last sinus lavage and the mucosa of right maxillary sinus (tested side) and left side (control) were collected for histopathological evaluation. Results: The sinonasal exposure to SEB resulted in epithelial thickening, inflammatory cells infiltration (tissue eosinophilia and neutrophilia) and reduction of ciliated cells. The exposure to LPS resulted in epithelial and subepithelial thickening, inflammatory cells infiltration, epithelial and subepithelial eosinophilia and increased subepithelial fibrosis. The exposure to LTA resulted in epithelial and subepithelial thickening, subepithelial inflammatory cells infiltration and eosinophilia and increased subepithelial fibrosis. Conclusion: This study reported the effects of bacterial toxins on the the sinonasal mucosa of ovalbumin-sensitized rabbits, demonstrating similar changes that are observed in CRS patients. Our results show that this is a viable animal model of CRS. Further studies are need to elucidate whether the pathomechanisms in this model are similar to what are observed in humans.

No description available.

La vaccination muqueuse est un moyen efficace de lutter contre les pathogènes qui utilisent les muqueuses comme porte d’entrée. Cependant, la vaccination muqueuse avec des antigènes non réplicatifs nécessite l’utilisation d’adjuvants. Les molécules de la famille de la toxine du choléra, l’entérotoxine thermolabile d’E.coli (LT), la toxine du choléra (CT) ainsi que le mutant LT-R192G et les sous-unités B non toxiques de ces toxines (LTB et CTB) ont été montrées augmenter les réponses immunitaires contre des antigènes coadministrés par voie muqueuse. Cependant leur mécanisme d’action est complexe et reste encore mal connu et des différences entre molécules entières et sous-unités B ont été rapportées ainsi que, pour une même molécule, des différences selon le modèle utilisé. Dans ce travail, nous avons étudié les effets de ces cinq molécules sur les réponses anticorps ainsi que sur les lymphocytes T CD4 dans un modèle murin d’immunisation intrarectale avec des pseudoparticules virales de rotavirus (VLP-2/6). Chez les souris non immunisées, nous avons montré que ces molécules, à l’exception de la CTB, diminuent in vitro les lymphocytes T régulateurs naturels CD4+CD25+Foxp3+, probablement par un mécanisme d’apoptose. Chez les souris immunisées, toutes les molécules étudiées induisent une même réponse anticorps sérique et fécale spécifique des VLP-2/6, qu’il s’agisse des molécules entières connues pour leur fort pouvoir adjuvant ou des sous-unités B qui, elles, ont été rapportées avoir un plus faible effet adjuvant voire un effet tolérogène dans certaines études. Concernant la réponse T CD4, les réponses spécifiques de l’antigène et de l’adjuvant ont été analysées. Des différences importantes ont été mises en évidence entre ces molécules. Notamment, seules les molécules entières (LT, LT-R192G et CT) induisent la production d’IL-2 et l’activation de lymphocytes T CD4+CD25+Foxp3- mémoires spécifiques de l’antigène tout en permettant la mise en place d’une régulation médiée par des lymphocytes T régulateurs CD4+CD25+Foxp3+ (boucle d’autorégulation), qui pourraient jouer un rôle majeur lors d’une réponse secondaire, afin d’éviter les réactions inflammatoires délétères. Malgré ces différences, toutes les molécules étudiées induisent la production d’IL-17, suggérant le rôle majeur de cette cytokine dans l’effet adjuvant.L’influence de la voie d’administration sur ces effets est en cours d’étude grâce à la comparaison avec la voie intranasale / Mucosal immunization is an important goal of vaccine development to protect against pathogens that use mucosa as portals of entry. However, the use of non-replicating antigens requires the addition of adjuvants.Cholera-like enterotoxins, cholera toxin (CT) from Vibrio cholerae and the heat-labile enterotoxin (LT) from toxinogenic strains of E. coli, as well as the mutant LR-192G and their B subunits (CTB and LTB) have been shown to increase immune responses against unrelated co-administered antigens by mucosal routes. However, their mechanism of action is very complex and not completely understood and differences exist between holotoxins and B subunits and within molecules, differences exist between the models used.In this work, we have studied the effects of these five molecules on antibody responses and on CD4+ T cell responses in a murine model of intrarectal immunization using rotavirus-like particles (2/6-VLP). In non-immunized mice, we have shown that all molecules, except CTB, decreased CD4+CD25+Foxp3+ natural regulatory T cells, probably by induction of apoptosis.In immunized mice, all molecules induced similar VLP-2/6 specific systemic and fecal antibody responses, teither he holotoxins, which are well known for their strong adjuvanticity or their B subunits with a less strong adjuvanticity but with also a tolerogenic effect in some studies.Regarding the CD4+ T cell response, antigen- and adjuvant- specific responses have been analysed. Important differences have been highlighted between the molecules. Among others things, only whole toxins (LT, LT-R192G and CT) trigger IL-2 production and activation of antigen specific memory CD4+CD25+Foxp3- T cells and at the same time antigen specific CD4+CD25+Foxp3+ regulatory T cells are activated which may control the effector response (Feedback loop regulation) and avoid deleterious inflammation. In spite of these differences, all studied molecules triggered IL-17 production, suggesting the major role of this cytokine in adjuvanticity. We are currently comparing the intrarectal and intranasl routes in order to evaluate the role played by the route of immunisation in different effects of these molecules

<p>Staphylococcus aureus је често присутан у вимену крава где може да узрокује појаву субклиничких и клиничких маститиса. Због способности синтезе термостабилних стафилококних ентеротоксина представља чест узрок тровања храном код људи. Многи аутори бавили су се испитивањем патогености сојева Staphylococcus aureus са аспекта здравља вимена, као и испитивањем патогености сојева пореклом из стадних узорака млека или из производа од млека, али постоји мало података о патогености сојева Staphylococcus aureus изолованих из узорака млека узетих из појединих четврти вимена крава, као и о њиховој филогенетској сродности са изолатима пореклом од људи. Због тога, циљ истраживања у оквиру ове докторске дисертације, био је да се испита патогеност изолата Staphylococcus aureus пореклом из појединачних узорака млека и секрета вимена крава и изолата пореклом из брисева гуше људи, као и да се испита филогенетска сродност ових изолата.<br />На 46 газдинстава у централној Србији, Калифорнија маститис тестом идентификоване су краве са поремећеном секрецијом. Са две велике фарме у Војводини узети су узорци секрета из четврти вимена крава са клиничким маститисом. На Институту за јавно здравље Војводине у Новом Саду, узети су и брисеви гуше људи. Из наведених узорака коришћењем стандардних микробиолошких метода за изолацију и идентификацију, као и API Staph теста и доказивањем присуства nuc гена методом ланчане реакције полимеразе (PCR) 86 изолата потврђено је као Staphylococcus aureus. Из четврти вимена крава са субклиничким маститисом изоловано је 62 изолата, из четврти вимена крава са клиничким маститисом изоловано је 13 изолата, а из брисева гуше људи 11 изолата Staphylococcus aureus. За одређивање способности синтезе стафилококних ентеротоксина (SE) коришћен је VIDAS&reg; SET2 тест на принципу ELFA методе. Присуство гена за синтезу појединих SE, присуство гена за синтезу Пантон Валентин леукоцидина (PVL) и гена за резистенцију на метицилин доказано је PCR методом. Диск дифузионом методом по Кирби-Бауеру одређена је осетљивост изолата на антимикробне лекове и то на амоксицилин, амоксицилин/клавуланску киселину, ампицилин, бацитрацин, неомицин, новобиоцин, пеницилин, тетрациклин, триметоприм и триметоприм/сулфаметоксазол. Анализом нуклеотидних секвенци гена за синтезу стафилококног протеина А утврђена је филогенетска сродност изолата Staphylococcus aureus пореклом из млека крава оболелих од субклиничког и клиничког маститиса, као и изолата пореклом из брисева гуше људи.<br />Из 111 узорака млека узетих из четврти са позитивним CMT тестом Staphylococcus aureus изолован је из 62 узорка млека, што износи 55,86%. Од укупно 62 изолата Staphylococcus aureus пореклом из четврти вимена крава са поремећеном секрецијом, 5 (8,06%) синтетише стафилококне ентеротоксине и то само SECs, док 6 (54,54%) од 11 изолата Staphylococcus aureus пореклом од људи, синтетише стафилококне ентеротоксине и то и SECs и SEB. Присуство гена за синтезу Пантон-Валентин леукоцидина утврђено је код 5 (8,06%) од 62 изолата Staphylococcus aureus пореклом из четврти вимена крава са поремећеном секрецијом, као и код 7 (63,64%) од 11 изолата Staphylococcus aureus пореклом од људи. Изолати Staphylococcus aureus пореклом из вимена крава резистентни су на мањи број антимикробних лекова од изолата Staphylococcus aureus пореклом од људи на основу испитивања осетљивости на 10 антимикробних лекова. Свих 86 изолата Staphylococcus aureus су сензитивни на новобиоцин и триметоприм-сулфаметоксазол, док је 77 изолата (89,54%) резистентно<br />на бацитрацин, 24 (27,91%) на пеницилин, а 14 (16,28%) на ампицилин. Присуство гена за метицилинску резистенцију утврђено је код 2 (18,18%) од 11 изолата Staphylococcus aureus пореклом од људи и код 1 (1,33%) од 75 изолата Staphylococcus aureus пореклом из вимена крава. На основу испитивања филогенетске сродности нуклеотидних секвенци гена за синтезу стафилококног протеина А код 75 изолата пореклом из вимена крава и 11 изолата пореклом од људи, 2 изолата Staphylococcus aureus пореклом од људи могу се сматрати прецима свих осталих изолата. Подаци о нуклеотидним секвенцама изолата Staphylococcus aureus пореклом од крава депоновани су у GenBank и додељени су им приступни бројеви од KJ023978 до KJ024046.<br />Резултати истраживања указују на различита патогена својства изолата Staphylococcus aureus пореклом од крава са поремећеном секрецијом, због чега би њихово што раније откривање могло да има значајну улогу у очувању здравља вимена и у добијању квалитетног и здравствено безбедног млека.<br />Неопходна су даља истраживања како би се у потпуности разјаснио утицај изолата Staphylococcus aureus из вимена крава на безбедност млека и производа од млека, као и на здравље људи.</p> / <p>Staphylococcus aureus je često prisutan u vimenu krava gde može da uzrokuje pojavu subkliničkih i kliničkih mastitisa. Zbog sposobnosti sinteze termostabilnih stafilokoknih enterotoksina predstavlja čest uzrok trovanja hranom kod ljudi. Mnogi autori bavili su se ispitivanjem patogenosti sojeva Staphylococcus aureus sa aspekta zdravlja vimena, kao i ispitivanjem patogenosti sojeva poreklom iz stadnih uzoraka mleka ili iz proizvoda od mleka, ali postoji malo podataka o patogenosti sojeva Staphylococcus aureus izolovanih iz uzoraka mleka uzetih iz pojedinih četvrti vimena krava, kao i o njihovoj filogenetskoj srodnosti sa izolatima poreklom od ljudi. Zbog toga, cilj istraživanja u okviru ove doktorske disertacije, bio je da se ispita patogenost izolata Staphylococcus aureus poreklom iz pojedinačnih uzoraka mleka i sekreta vimena krava i izolata poreklom iz briseva guše ljudi, kao i da se ispita filogenetska srodnost ovih izolata.<br />Na 46 gazdinstava u centralnoj Srbiji, Kalifornija mastitis testom identifikovane su krave sa poremećenom sekrecijom. Sa dve velike farme u Vojvodini uzeti su uzorci sekreta iz četvrti vimena krava sa kliničkim mastitisom. Na Institutu za javno zdravlje Vojvodine u Novom Sadu, uzeti su i brisevi guše ljudi. Iz navedenih uzoraka korišćenjem standardnih mikrobioloških metoda za izolaciju i identifikaciju, kao i API Staph testa i dokazivanjem prisustva nuc gena metodom lančane reakcije polimeraze (PCR) 86 izolata potvrđeno je kao Staphylococcus aureus. Iz četvrti vimena krava sa subkliničkim mastitisom izolovano je 62 izolata, iz četvrti vimena krava sa kliničkim mastitisom izolovano je 13 izolata, a iz briseva guše ljudi 11 izolata Staphylococcus aureus. Za određivanje sposobnosti sinteze stafilokoknih enterotoksina (SE) korišćen je VIDAS&reg; SET2 test na principu ELFA metode. Prisustvo gena za sintezu pojedinih SE, prisustvo gena za sintezu Panton Valentin leukocidina (PVL) i gena za rezistenciju na meticilin dokazano je PCR metodom. Disk difuzionom metodom po Kirbi-Baueru određena je osetljivost izolata na antimikrobne lekove i to na amoksicilin, amoksicilin/klavulansku kiselinu, ampicilin, bacitracin, neomicin, novobiocin, penicilin, tetraciklin, trimetoprim i trimetoprim/sulfametoksazol. Analizom nukleotidnih sekvenci gena za sintezu stafilokoknog proteina A utvrđena je filogenetska srodnost izolata Staphylococcus aureus poreklom iz mleka krava obolelih od subkliničkog i kliničkog mastitisa, kao i izolata poreklom iz briseva guše ljudi.<br />Iz 111 uzoraka mleka uzetih iz četvrti sa pozitivnim CMT testom Staphylococcus aureus izolovan je iz 62 uzorka mleka, što iznosi 55,86%. Od ukupno 62 izolata Staphylococcus aureus poreklom iz četvrti vimena krava sa poremećenom sekrecijom, 5 (8,06%) sintetiše stafilokokne enterotoksine i to samo SECs, dok 6 (54,54%) od 11 izolata Staphylococcus aureus poreklom od ljudi, sintetiše stafilokokne enterotoksine i to i SECs i SEB. Prisustvo gena za sintezu Panton-Valentin leukocidina utvrđeno je kod 5 (8,06%) od 62 izolata Staphylococcus aureus poreklom iz četvrti vimena krava sa poremećenom sekrecijom, kao i kod 7 (63,64%) od 11 izolata Staphylococcus aureus poreklom od ljudi. Izolati Staphylococcus aureus poreklom iz vimena krava rezistentni su na manji broj antimikrobnih lekova od izolata Staphylococcus aureus poreklom od ljudi na osnovu ispitivanja osetljivosti na 10 antimikrobnih lekova. Svih 86 izolata Staphylococcus aureus su senzitivni na novobiocin i trimetoprim-sulfametoksazol, dok je 77 izolata (89,54%) rezistentno<br />na bacitracin, 24 (27,91%) na penicilin, a 14 (16,28%) na ampicilin. Prisustvo gena za meticilinsku rezistenciju utvrđeno je kod 2 (18,18%) od 11 izolata Staphylococcus aureus poreklom od ljudi i kod 1 (1,33%) od 75 izolata Staphylococcus aureus poreklom iz vimena krava. Na osnovu ispitivanja filogenetske srodnosti nukleotidnih sekvenci gena za sintezu stafilokoknog proteina A kod 75 izolata poreklom iz vimena krava i 11 izolata poreklom od ljudi, 2 izolata Staphylococcus aureus poreklom od ljudi mogu se smatrati precima svih ostalih izolata. Podaci o nukleotidnim sekvencama izolata Staphylococcus aureus poreklom od krava deponovani su u GenBank i dodeljeni su im pristupni brojevi od KJ023978 do KJ024046.<br />Rezultati istraživanja ukazuju na različita patogena svojstva izolata Staphylococcus aureus poreklom od krava sa poremećenom sekrecijom, zbog čega bi njihovo što ranije otkrivanje moglo da ima značajnu ulogu u očuvanju zdravlja vimena i u dobijanju kvalitetnog i zdravstveno bezbednog mleka.<br />Neophodna su dalja istraživanja kako bi se u potpunosti razjasnio uticaj izolata Staphylococcus aureus iz vimena krava na bezbednost mleka i proizvoda od mleka, kao i na zdravlje ljudi.</p> / <p>Staphylococcus aureus is often present in cow&#39;s udder and may cause the occurrence of subclinical and clinical mastitis. Due to the ability to synthesize thermostable staphylococcal enterotoxins, it is a common cause of food poisoning in humans. Many authors have dealt with the examination of the pathogenicity of strains of Staphylococcus aureus in terms of udder health, as well as examining the pathogenicity of strains originating from bulk tank milk samples or dairy products, but there is a scarce amount of data on the pathogenicity of strains of Staphylococcus aureus originating from individual samples from udder quarters, as well as on their phylogenetic relatedness with isolates from humans. Therefore, the goal of the research within this dissertation was to investigate the pathogenicity of Staphylococcus aureus originating from individual samples of milk and udder secretions, as well as isolates from throat swabs of people and to examine the phylogenetic relatedness of these isolates.<br />On 46 dairy farms in central Serbia, cows with abnormal secretion were identified using California mastitis test. From two large dairy farms in Vojvodina samples were taken from udder quarters with clinical mastitis. Human throat swabs were collected at the Institute of Public Health of Vojvodina in Novi Sad. From these samples a total of 86 strains of Staphylococcus aureus were isolated and confirmed by standard microbiological methods for isolation and identification, as well as API Staph test and by detection of nuc gene using the Polymerase Chain Reaction (PCR). 62 isolates of Staphylococcus aureus were isolated from udder quarters suffering subclinical mastitis, 13 isolates were isolated from udder quarters suffering clinical mastitis, and 11 isolates of Staphylococcus aureus were isolated from human throat swabs. VIDAS &reg; SET2 was used for determining the ability of the synthesis of staphylococcal enterotoxins (SE) by the ELFA method and PCR method for the presence of respective SE genes. PCR method was used for determining the presence of gene for the synthesis of the Panton Valentine leukocidin (PVL), and the gene responsible for methicillin resistance in strains of Staphylococcus aureus. Susceptibility to antimicrobial drugs was determined by disk diffusion method by Kirby - Bauer to amoxicillin, amoxicillin/ clavulanic acid, ampicillin, bacitracin, neomycin, novobiocin, penicillin, tetracycline, trimethoprim and trimethoprim/sulfamethoxazole. Phylogenetic relatedness was determined by analyzing the nucleotide sequences of the genes for the synthesis of staphylococcal protein A between isolates of Staphylococcus aureus originating from cow&#39;s udder quarters suffering subclinical and clinical mastitis, as well as isolates of Staphylococcus aureus originating from human throat swabs.<br />Out of 111 milk samples from CMT positive udder quarters, Staphylococcus aureus was isolated from 62 samples, which amounts to 55.86%. Out of 62 isolates of Staphylococcus aureus originating from udder quarters with abnormal secretion, 5 (8.06%) synthesized by staphylococcal enterotoxin and only it was SECs, while 6 (54.54%) out of 11 isolates of Staphylococcus aureus originating from humans, synthesize staphylococcal enterotoxin and they were SECs and SEB. The presence of a gene for the synthesis of the Panton - Valentine leukocidin was determined in 5 (8.06%) out of 62 Staphylococcus aureus strains originating from the udder quarters with abnormal secretion, and in 7 (63.64%) out of 11 strains of Staphylococcus aureus origin of humans. Isolates of Staphylococcus aureus originating from cow&#39;s udder were<br />resistant to fewer antimicrobials than isolates of Staphylococcus aureus originating from humans on the basis of susceptibility to 10 antimicrobials. All 86 strains of Staphylococcus aureus were sensitive to novobiocin and trimethoprim/ sulfamethoxazole, 77 strains (89.54%) were resistant to bacitracin, 24 (27.91%) to penicillin, and 14 (16.28%) to ampicillin. The presence of the gene for methicillin resistance was found in 2 (18.18%) out of 11 isolates of Staphylococcus aureus originating from humans and in 1 (1.33%) out of 75 isolates of Staphylococcus aureus originating from the cow&rsquo;s udder. Analyzing the phylogenetic relatedness based on nucleotide sequences of the genes for the synthesis of staphylococcal protein A from 75 isolates from cow&#39;s udder, and 11 isolates from humans, 2 isolates of Staphylococcus aureus originating from humans can be considered to be the ancestors of all the other isolates in terms of gene mutation ratio give at respective period. Data on the nucleotide sequences of isolates of Staphylococcus aureus originating from cows were deposited in GenBank and assigned the accession numbers of KJ023978 to KJ024046.<br />Results of study on diverse pathogenic properties of Staphylococcus aureus originating from cows with abnormal secretion, indicate the importance of their early detection that could have a significant role in maintaining the cow&rsquo;s udder health and in obtaining quality and safe milk.<br />Further research is necessary in order to fully clarify the impact of Staphylococcus aureus from the cow&#39;s udder to the safety of milk and dairy products, as well as human health.</p>