Orientador: Brenda Paula Figueiredo de Almeida Gomes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T13:34:38Z (GMT). No. of bitstreams: 1 Duque_ThaisMageste_M.pdf: 4524163 bytes, checksum: caf90cf8c083cc156cb48692e4b59821 (MD5) Previous issue date: 2013 / Resumo: A doença periodontal crônica atinge as estruturas de suporte dos dentes e é uma das causas mais importantes da perda dos dentes em adultos. Essa doença de longa duração que não regride com a terapia, já pode ter causado uma alteração irreversível na polpa, mesmo quando clinicamente, responde de forma positiva aos testes de sensibilidade pulpar. Os lipopolissacarideos (LPS) presente nas paredes das bactérias Gram negativas e citocinas pró inflamatórias funcionam como fatores estimulantes para a resposta imune, causando destruição óssea e exacerbando o processo inflamatório. Assim, o objetivo do presente estudo foi avaliar a influência do tratamento endodôntico em pacientes com doença periodontal crônica, através da análise microbiológica por pcr, quantificação de endotoxinas e monitoramento de citocinas pró inflamatórias em bolsas periodontais e canais radiculares. Foram selecionados 15 dentes com envolvimento periodontal crônico e sensibilidade pulpar positiva. Após o preparo químico mecânico (PQM), os dentes foram divididos em três grupos: G1- sessão única (n=5); GII - Ca(OH)2 associado à clorexidina gel 2% (CHX-gel) (n=5); GIII - Ca(OH)2 + Solução salina (SS). Amostras foram coletadas em três diferentes momentos da terapia endodôntica: inicial, após PQM e após medicação intracanal (MIC) por 30 dias, através de cones de papéis apirogênicos/estéreis. Os microrganismos foram identificados através da técnica molecular do PCR simples (16S rDNA) com o uso de primers específicos para Fusobacterium nucleatum, Prevotella intermédia, complexo vermelho (Porphyromonas gingivalis, Tannerella forsythia e Treponema denticola), Gemella morbillorum e Parvimonas micra. Para quantificação de endotoxina foi realizado o teste Lisado de Amebócito Limulus (LAL) e, para a quantificação de citocinas IL1 ?, IL1 ?, TNF ? e PGE2, foi utilizado o ensaio imunoenzimático ELISA. Os resultados mostraram que após 1 ano, dos 15 dentes tratados, 5 foram extraídos. Os outros 10 dentes apresentaram melhora no quadro clínico relacionada à profundidade de sondagem e mobilidade. Nas bolsas periodontais iniciais, pelo menos um dos microrganismos do complexo vermelho estiveram presentes em 12/15 amostras e os 3 juntos estiveram presentes em 6/15. Amostras do canal inicial mostraram a presença de Parvimonas micra e Fusobaterium nucleatum detectadas em 80% dos casos. Não houve associação entre as bactérias encontradas inicialmente nas bolsas e nos canais radiculares. Após o PQM e uso de MIC por 30 dias, houve redução do conteúdo microbiano em ambos os sítios. A concentração de endotoxinas nas bolsas periodontais iniciais foram altas (192,81 EU/mL), mas após o PQM, uma redução (19,65 EU/mL) significativamente estatística foi observada. Os canais radiculares apresentaram concentrações de endotoxinas quase nulas (0,1 EU/mL), sendo compatível com o estado pulpar e assim se manteve durante as outras coletas. Avaliando as medicações testadas, a redução de LPS nas bolsas apresentou melhores resultados com o uso do Ca(OH)2 associado à solução salina (p< 0,05). Houve redução de todas as citocinas pró inflamatórias, quando comparadas às coletas iniciais e após o uso da medicação das bolsas e dos canais, sendo esta estatisticamente significativa em relação a IL1? e TNF?. Não houve correlação positiva entre citocinas e endotoxinas nas bolsas periodontais. Conclui-se que o complexo vermelho está presente na doença periodontal e que a presença de uma medicação intracanal diminui a concentração do LPS e de citocinas inflamátorias nos canais radiculares e bolsas periodontais / Abstract: The chronic periodontal disease affects the supporting structures of the teeth and is a major cause of tooth loss in adults. This long-term illness that does not regress with therapy, may have already caused an irreversible change in the pulp, even when clinically responds to pulp sensitivity tests. The lipopolysaccharide (LPS) present in the walls of gram negative bacteria and inflammatory cytokines act as factors for stimulating the immune response, causing bone destruction and increasing inflammation. The objective of this study was to evaluate the influence of endodontic treatment in patients with chronic periodontitis using microbiological analysis by pcr, endotoxin quantification and monitoring of inflammatory cytokines in periodontal pockets and root canals. Samples were taken from 15 teeth with chronic periodontal involvement and positive pulp sensitivity was selected. After chemical mechanical preparation (MCP), the teeth were divided into three groups: G1- one visit (n = 5), GII - Ca(OH)2 associated with chlorhexidine gel 2% (CHX-gel) (n = 5) ; GIII - Ca(OH)2 + saline solution (SS). Samples were collected at three different times in endodontic therapy: initial, after MCP and after intracanal medication (ICM) for 30 days, through paper cones apirogênics / sterile. The microorganisms were identified by PCR simple molecular technique (16S rDNA) by using specific primers for Fusobacterium nucleatum, Prevotella intermédia, red complex (Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia), and Parvimonas micras. For quantification of endotoxin was performed the Limulus Amebocyte Lysate (LAL) test and for the quantification of cytokines IL-1 ?, IL-1 ?, TNF ? and PGE2 was used ELISA immunoenzymatic assay. The results showed that after 1 year, 15 of the treated teeth, 5 were extracted. The other 10 teeth showed improvement related to probing depth and mobility. In the initial periodontal pockets, at least one of the microorganisms of the red complex were present in 12/15 samples and 3 together were present in 6/15. Canals samples showed the presence of Parvimonas micra and Fusobaterium nucleatum detected in 80% of cases. There was no association between the bacteria found in the pockets and root canals. After the CMP and use of ICM for 30 days, there was reduction of microbial content in both places. The initial endotoxin concentration in periodontal pockets were high (192.81 EU / ml), but after the CMP the reduction (19.65 EU / ml) was statistically significantly observed. Root canals showed low concentrations of endotoxin (0.1 EU / mL), being compatible with state of the pulp and maintained for other samples. Accordin the tested medications, reduction of LPS in the pockets showed better results with the use of Ca(OH)2 associated to saline solution (p <0.05). There was a reduction of all inflammatory cytokines when compared to the initial samples and after use of the dressing in pocktes and canals, being statistically significant in relation to IL1? and TNFa. There was no positive correlation between endotoxin and cytokines in periodontal pockets. In conclusion the red complex is present in the periodontal disease and that the presence of dressing decreases the concentration of inflammatory cytokines and LPS in root canals and periodontal pockets / Mestrado / Endodontia / Mestra em Clínica Odontológica

Les systèmes Toxine Antitoxine (TA) sont présents chez la plupart des génomes bactériens. Nous rapportons dans cette étude la présence de tels systèmes chez la bactérie pathogène Helicobacter pylori. Ce nouveau système TA de type I, de la même manière que les autres systèmes de type I décrits, est composé d’une toxine peptidique membranaire (AapA) dont la traduction est inhibée par un ARNnc (IsoA) suivant une interaction côté 5’ non traduit de l’ARNm. La structuration particuliere de l’ARNm a permit l’identification d’orthologue de ce système dans les chromosomes des genres Helicobacter et Campylobacter mais aussi sur leur patrimoine génétique mobiles, tels les plasmides. Ceci impliquant leur potentielle acquisition dans le génome par transfert génétique horizontal. La deuxième partie de l’étude se focalise sur la résolution par RMN du liquide de la structure atomique de la toxine AapA1 dans un environnement pseudo-membranaire. Une approche par mutation révèle les determinants structuraux de la toxicité, pointant la présence d’une empreinte de charge dans la partie helicoidale transmenbranaire de la toxine présente aussi chez d’autres toxines de Type I. La dernière partie de l’étude se centre sur l’expression et la purification de la toxine afin d’en étudier la structure en environnement membranaire complexe par RMN du solide. Les résultats prometteurs ouvrent la voie à une caractérisation de la toxine par l’expérience de PISEMA. / Toxin-antitoxin (TA) systems are present in almost all bacterial genomes. Here we report that the genome of the major human gastric pathogen, Helicobacter pylori, is hosting several copies of a new family of type I TA systems. Similarly to other type I TA systems, the toxin (AapA) is a small membrane protein whose expression is controlled by a small antisense RNA (IsoA, antitoxin) that binds to the 5’ untranslated region (UTR) of the mRNA. In addition we used the strong conservation of the mRNA folding to identify homologs of this TA system not only in other Helicobacter and Campylobacter chromosomes but also on plasmids, indicating that this new TA system might have been spread over different genomes via horizontal gene transfer.The second part of the study take account of the AapA toxin itself. We acutely determine the structure of AapA1 by liquid state NMR in membrane mimicking environment. We then probe first structural insight on atomic structural determinants of its toxicity following a mutation studies.These results reveal a particular charge pattern on the transmembrane α helix domain of AapA toxin similary to other Type I toxin. A third part of the studies is based on expression and purification of the toxin in order to determine the structure in complex membrane environment by solid state NMR. The promosisng result open the way to characterize the toxin by PISEMA experiment.

Sub-acute/chronic inflammatory diseases are often associated with altered inflammatory response, leading to increased host vulnerability to secondary inflammatory challenges. In the first study, by employing streptozotocin (STZ)-induced diabetes in mice, we further investigate mechanisms leading to enhanced polymorphonuclear leukocytes (PMN) response under hyperglycemia. We show that existence of a proinflammatory state associated with broad increases of macrophages in various organs plays a dominant role in promoting PMN response in diabetic mice. Studies of PMN infiltration during zymosan-induced peritonitis reveal that hyperglycemia enhances PMN recruitment through increasing F4/80+ macrophages in the peritoneal cavity. Insulin reversal of hyperglycemia reduces peritoneal macrophage numbers and ameliorates PMN infiltration. Significantly increased macrophages are also observed in the liver, kidneys, and intestines under hyperglycemia, and are attributable to exacerbated nephropathy and colitis when respective inflammatory conditions are induced. We also find that significant monocytosis of inflammatory F4/80+Gr-1+ monocytes from the spleen and macrophage proliferation in situ synergistically contribute to the increased macrophage population under hyperglycemia. In conclusion, our results demonstrate that STZ-induced hyperglycemic/diabetic mice develop a systemic proinflammatory state mediated by broad infiltration of macrophages. In the second study, we focus on the identification of the carrier that binds to and delivers Shiga toxin 2(Stx2) to the target organ causing hemolytic uremic syndrome (HUS). By employing a murine HUS model through co-injection of LPS-Stx2, we show that, adoptive transfer of CD11b+ leukocytes, but not CD11b- leukocytes, RBC, platelets or plasma, isolated from mice with HUS induces HUS in healthy recipients. Interestingly, we find that LPS priming of mice significantly promotes CD11b+ leukocytes binding to Stx2. Compared to CD11b+ leukocytes from mice without LPS priming, CD11b+ leukocytes isolated from mice after LPS priming demonstrate higher frequencies of toxin binding and augmented potency to induce HUS. In sum, our results demonstrate peripheral CD11b+ myeloid leukocytes act as effective Stx2 carriers that deliver toxin to kidneys causing HUS and that LPS-induced inflammation enhances the carrier capacity and aggravates HUS.

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.

Les systèmes toxine-antitoxine (STA) sont généralement constitués de deux petites protéines cytoplasmiques : une toxine stable et une antitoxine instable capable de neutraliser la toxine et de réprimer l’expression de l’opéron toxine-antitoxine. Une étude menée au laboratoire avait mis en évidence que la perte du gène hicB3 (ypo3369) de Y. pestis, codant une antitoxine solitaire putative, entraine un retard de la croissance bactérienne in vitro et une atténuation de la virulence dans un modèle murin de peste bubonique (Pradel et al., 2014). Par analyse in silico, nous avons détecté, en amont de hicB3, un petit gène non annoté candidat pour coder la toxine HicA3. La surproduction de HicA3 provoque la bactériostase chez Escherichia coli et Y. pestis et la production subséquente de HicB3 restaure la croissance. HicA3 et HicB3 constituent donc un STA fonctionnel. Cependant, la perte du STA HicA3B3 n’affecte pas la virulence de Y. pestis dans un modèle murin de peste bubonique. Nous avons ensuite purifié et caractérisé les protéines HicA3 et HicB3. La toxine HicA3 est une ribonucléase monomérique de 66 aa qui comporte un résidu histidine catalytique essentiel pour son activité. L’antitoxine HicB3 a une double fonction : elle interagit avec HicA3 pour la neutraliser et elle réprime le promoteur de l’opéron hicA3B3. Des expériences de retard sur gel et de fusions transcriptionnelles avec un gène rapporteur ont révélé que l’antitoxine HicB3 et le complexe HicA3-HicB3 se fixent sur deux opérateurs chevauchant les boîtes -10 et -35 du promoteur PhicA3. Nous avons également résolu la structure cristalline de l’antitoxine HicB3 et celle du complexe HicA3-HicB3. HicB3 est un tétramère qui comporte deux domaines de fixation à l’ADN du type ruban-hélice-hélice et deux domaines de neutralisation de la toxine. / Toxin-antitoxin systems (TAS) are generally constituted by two small cytoplasmic proteins: a stable toxin and an unstable antitoxin which neutralizes the toxin and represses the expression of the toxin-antitoxin operon. In previous research, our lab found that Yersinia pestis lacking the hicB3 (ypo3369) gene, encoding a putative orphan antitoxin, has a growth defect in vitro and is attenuated for virulence in a murine model of bubonic plague (Pradel et al., 2014). In silico analysis revealed a small gene upstream of hicB3, encoding a putative toxin that we called HicA3. HicA3 overproduction generates bacteriostasis of Escherichia coli and Y. pestis, and the subsequent production of HicB3 restores cell growth. HicA3 and HicB3 thus constitute a functional TAS. However, the lack of the HicA3B3 TAS does not affect Y. pestis virulence in a murine model of bubonic plague. We then purified and characterized the HicA3 and HicB3 proteins. The HicA3 toxin is a monomeric 66-aa ribonuclease with a catalytic histidine residue required for its activity. The HicB3 antitoxin has two functions: it binds and neutralizes HicA3 and it represses the hicA3B3 operon promoter. Gel-shift assays and transcriptional reporter fusion experiments showed that both HicB3 and the HicA3-HicB3 complex bind to two operators overlapping the -10 and -35 boxes of the PhicA3 promoter. We also solved the crystal structures of the HicB3 antitoxin and the HicA3-HicB3 complex. HicB3 is a tetramer with two DNA binding domains of the ribbon-helix-helix type and two toxin neutralization domains.

No description available.

Nonmenstrual toxic shock syndrome (TSS) due to Staphylococcus aureus can lead to significant morbidity and mortality. While drotrecogin alfa (DA) has been employed in patients with Methicillin-resistant Staphylococcus aureus (MRSA) severe sepsis and septic shock, its utility in TSS remains unclear. The authors report a case of severe sepsis in the setting of MRSA-associated TSS that responded to treatment with DA. This case illustrates a potential role for DA in the treatment of toxic shock syndromes and emphasizes the importance of aggressive diagnostic and therapeutic modalities in approaching these conditions.

Post-thoracotomy pain syndrome (PTPS) is a traumatic neuropathy that can affect as many as 50% of patients undergoing thoracotomy. Patients are often refractory to conservative management and may require multiple analgesics for adequate pain control. Botulinum toxin, derived from Clostridium botulinum, has many uses in treating conditions involving spasticity, dystonia, chronic migraine, and a variety of pain disorders including neuropathies. Botulinum toxin type A injections may provide an alternative or adjunct to improve symptom management in patients with PTPS.

No description available.

AB-type protein toxins such as cholera toxin (CT) consist of a catalytic A subunit and a cell-binding B subunit. CT proceeds through the secretory pathway in reverse, termed retrograde trafficking, and is delivered to the endoplasmic reticulum (ER). In order for the catalytic A1 subunit to become active it must separate from the rest of the holotoxin, and this dissociation event occurs in the ER lumen. CTA1 assumes an unfolded conformation upon dissociation from the holotoxin and is recognized by ERassociated degradation (ERAD), a quality control system that recognizes and exports misfolded proteins to the cytosol for degradation by the 26S proteasome. CTA1 is not degraded by the 26S proteasome because it has few sites for poly-ubitiquination, which is recognized by the cap of the 26S proteasome for degradation. Thus, CTA1 escapes the degradation of ERAD while at the same time using it as a transport mechanism into the cytosol. It was originally proposed that CTA1 is thermally stable and that ER chaperones actively unfolded CTA1 for translocation to the cytosol. In contrast, we hypothesized that the dissociated CTA1 subunit would unfold spontaneously at 37°C. This study focused on the three conditions linked to CTA1 instability and translocation: (i) CTA1 dissociation from the holotoxin, (ii) the translocation-competent conformation of CTA1, and the extraction of CTA1 from the ER into the cytosol. Disruption of any of these events will confer resistance to the toxin. The original model suggested that PDI actively unfolds CTA1 to allow for translocation. However, Fourier transform infrared iv spectroscopy (FTIR) and surface plasmon resonance (SPR) data we have gathered demonstrated that PDI dislodges CTA1 from the rest of the holotoxin without unfolding CTA1. Once released by the holotoxin, CTA1 spontaneously unfolds. PDI is thus required for the toxicity of CT, but not as an unfoldase as originally proposed. CTA1 must maintain an unfolded conformation to keep its translocation-competent state. Based on our model, if CTA1 is stabilized then it will not be able to activate the ERAD translocation system. Our SPR and toxicity results demonstrated that treatment with 4- phenylbutyrate (PBA), a chemical chaperone, stabilizes the structure of CTA1. This stabilization resulted in a decrease in translocation from the ER to the cytosol and a block of intoxication, which makes it a viable candidate for a therapeutic. Because CTA1 exits the ER in an unfolded state, there must be a driving force for this translocation. We hypothesized that Hsp90, a cytosolic chaperone, is responsible for the translocation of CTA1 across the membrane. Previous research had shown Hsp90 to be present on the cytosolic face of the ER and had also shown that Hsp90 will refold exogenously added proteins that enter the cytosol. Using drug treatments and RNAi, we found that Hsp90 is required for the translocation of CTA1 from the ER lumen to the cytosol, a brand new function for this chaperone. We have provided evidence to support a new, substantially different model of CTA1 translocation. CTA1 does not masquerade as a misfolded protein in order to utilize ERAD for entry into the cytosol; it actually becomes misfolded and is treated as any other ERAD substrate. The spontaneous unfolding of CTA1 is the key to its v recognition by ERAD and ultimately its translocation into the cytosol. Host factors play very important roles in intoxication by AB toxins and are targets for blocking intoxication.