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A Febre Reumática, complicação tardia de uma infecção de orofaringe causada pelo Streptococcus pyogenes (estreptococo -hemolítico do grupo A de Lancefield), tem como conseqüência a Cardiopatia Reumática, explicada pelo mimetismo molecular entre proteínas cardíacas humanas e a associação de proteínas e carboidratos da membrana do S. pyogenes. A profilaxia secundária com a PGB 1.200.000 UI IM propõe-se a evitar novos surtos, sendo administrada em intervalos de vinte e um dias nos países com alto índice de estreptococcia. A lesão valvar predispõe à Endocardite Infecciosa, que resulta de bacteriemias causadas por focos infecciosos de origem bucal em cerca de 40% dos casos. Os Streptococcus Viridans constituem o grupo mais comumente encontrado nas Endocardites Infecciosas, em especial os Streptococcus sanguinis e Streptococcus oralis. O efeito do uso crônico da PGB não foi estudado com especificidade para essa microbiota. Assim, foi avaliada, qualitativa e quantitativamente, a microbiota bucal de 100 pacientes, aos 7 e 21 dias, após profilaxia secundária para a Febre Reumática com a PGB 1.200.000 UI IM e comparada com a de 100 pacientes portadores de doença arterial coronariana sem antecedentes de Febre Reumática. As espécies avaliadas foram divididas em S. sanguinis, S. oralis e outras espécies de Streptococcus Viridans Foram coletadas amostras de saliva pela mastigação de goma de parafina e transportadas em meio VMGA II S. As culturas foram semeadas em ágar Columbia CNA com 5% de sangue desfibrinado puro de carneiro com acréscimo de penicilina G. e incubadas a 35ºC em estufa de CO2 por 72 horas. As colônias sugestivas de Streptococcus foram submetidas a testes bioquímicos para confirmação de gênero e espécie. A concentração inibitória mínima foi determinada pelo método Etest e interpretada segundo os padrões do Clinical and Laboratory Standards Institute. Não houve diferença quanto à presença do S. sanguinis nos grupos estudados (P=0,40). O S. oralis prevaleceu aos 7 dias de PGB em relação ao grupo controle (P=0,01). Quanto à identificação de outras espécies, houve maior número de cepas nos pacientes do grupo controle quando comparados aos do grupo de estudo aos 7 e 21 dias de PGB (P<0,001). Os números de UFC/ml de S. sanguinis, S. oralis e de outras espécies foram comparados entre os grupos e não houve diferença entre eles (P=0,96; P=0,60 e P=0,77; respectivamente). Quanto às CIM do S. sanguinis e do S. oralis, não houve diferença entre os grupos (P=0,79 e P=0,13; respectivamente). Todos os testes estatísticos foram realizados em um nível de significância de 5%. Concluiu-se que o S. oralis prevaleceu aos 7 dias de PGB 1.200.000 UI IM; os Streptococcus Viridans de outras espécies prevaleceram no grupo controle; o número de UFC/mL de saliva não diferiu nos grupos estudados, a susceptibilidade dos S. sanguinis e S. oralis à penicilina G não foi alterada pela ação da PGB 1.200.000 UI IM a cada 21 dias e, por fim, a PGB não provocou reações de hipersensibilidade em nenhum paciente do estudo / Rheumatic fever is the result of a Streptococcus pyogenes (group A -hemolytic Streptococcus) infection of the upper respiratory tract. Rheumatic heart disease is a rheumatic fever consequence and is elucidated by the molecular mimicry between human cardiac proteins and group A streptococcal proteins and carbohydrates association. The secondary prophylaxis with 1,200,000 U BPG every three weeks is used for prevention of recurrent rheumatic fever in developing countries. Valvar defects are a risk for infective endocarditis which is resulted of bacteriemia caused for oral infectious focuses in 40% of cases. Viridans streptococci are the predominant group recovered in infective endocarditis, specially Streptococcus sanguinis and Streptococcus oralis. The effect of chronic BPG wasnt studied with specificity to these pathogens yet. Therefore, the oral microbiota was evaluated, qualitatively and quantitatively, at 7 and 21 days after secondary prophylaxis with BPG to rheumatic fever (study group), in a hundred patients and in comparison to another hundred patients with coronary heart disease who never acquired rheumatic fever (control group). The species evaluated were divided in S. sanguinis, S. oralis and another Streptococcus species. It was collected samples of chewing-stimulated saliva (1ml) and transported in VMGA II S medium. The samples were cultured in pure and with penicillin G 5% sheep blood Columbia ágar (CNA), incubated for 72 hours in an atmosphere containing 5% CO2 at 35ºC. The strains that were suggestive to Streptococcus were identified by biochemical tests to confirm bacteria species and genus. Minimal inhibitory concentration was determined by Etest method and interpreted in accordance to Clinical and Laboratory Standards Institute. The results showed that there was no difference in S. sanguinis presence in all groups (P=0.40). S. oralis prevailed in 7 days BPG group in comparison to control group (P=0.01). The control group showed the highest number of others species in comparison to 7 and 21 days BPG (P<0.001). CFU/ml numbers of S. sanguinis, S. oralis and other species strains were compared in 7 and 21 days BPG to control group and there was no difference among themselves (P=0.96, P=0.60 and P=0.77; respectively). There was no difference in S. sanguinis and S. oralis MICs among the study and control groups (P=0.79 and P=0.13). All statistic tests were done at 5% significance level. It was concluded that S. oralis prevailed in 7 days BPG group in comparison to control group; other species of Viridans streptococci prevailed in control group. The number of CFU/mL did not differ in both studied groups; the penicillin susceptibility of S. sanguinis and S. oralis did not change by BPG every three weeks and, by the end, it was not observed hypersensitivity reactions to penicillin in neither of the patients of this study

A penicilina G é um dos antibióticos mais importantes. Além de possuir um baixo preço e comprovada eficácia de tratamento, mostra inúmeras possibilidades para a redução da morbidade e mortalidade por doenças infecciosas em todo o mundo. Como eventualmente este medicamento causa sequelas no parênquima renal e estruturas associadas, e sendo que a secreção da rede tubular renal contribui para a excreção da penicilina G, onde cerca de 60% do antibiótico é eliminado pela urina, nos propomos a fazer um estudo das principais alterações que possam ocorrer na pelve renal de ratos normais e ratos induzidos à diabetes. Este projeto tem o propósito de descrever e analisar as fibras colágenas, musculares lisas e elásticas da pelve renal de ratos wistar observando alterações estruturais e ultraestruturais dos grupos experimentais quando comparados ao grupo controle com relação ao uso da penicilina G. Os ratos foram divididos em 4 grupos, ratos Wistar normais (N); ratos Wistar tratados com penicilina G (NP); ratos Wistar induzidos à diabetes (D); ratos Wistar diabéticos com penicilina G (DP). Os ratos dos grupos D e DP foram induzidos ao diabetes por aloxano. A região da pelve renal com representação das fibras foi coletada e reduzida em pequenos fragmentos. Os cortes obtidos foram utilizados para Microscopia Eletrônica de Transmissão e corados pelos seguintes métodos para Microscopia Óptica: Hematoxilina Férrica para evidenciação de fibras elásticas; Resorcina fucsina para evidenciação de fibras elásticas e elaunínicas; Resorcina fucsina após oxidação com solução aquosa a 1% de oxona para evidenciação de fibras elásticas, elaunínicas e oxitalânicas; Azan para evidenciação do componente colágeno e muscular lisa; Picrosírius para observação do componente colágeno (especificamente tipo I e III); e Hematoxilina e Eosina, para evidenciação do componente celular. A análise microscópica e a histomorfometria mostraram que a Penicilina G altera os componentes fibrosos da pelve renal, fazendo com que as áreas de fibras musculares lisas e de colágeno tipo III fossem aumentadas e as fibras elásticas maduras diminuídas (neste caso, apenas entre N e NP). O Diabetes mellitus mostrou-se como uma doença metabólica também capaz de alterar a morfologia da pelve, fazendo com que a área de fibras musculares lisas aumentasse, a área de colágeno tipo I e a quantidade de fibras elásticas maduras e elaunínicas diminuísse e as oxitalânicas aumentassem, além de um notável aumento na quantidade de mitocôndrias. Podemos inferir que a antibioticoterapia feita pela penicilina G e o diabetes, provocam diferenças estruturais e ultraestruturais na pelve renal dos ratos Wistar, principalmente na organização dos componentes fibrosos elástico, muscular e colágeno. / Penicillin G is the most important antibiotics. Besides having a low cost and proven effectiveness of treatment, it shows great possibilities for reducing morbidity and mortality from infectious diseases worldwide. As this medicine may cause sequelae in the renal parenchyma and associated structures, and since the net renal tubular secretion contributes to the excretion of penicillin G, where about 60% of the antibiotic is eliminated in urine, this study aims to investigate the main structural and ultrastructural changes occurring in the kidney of normal and diabetes rats. Thus, this project aims to describe and analyze the collagen fibers, smooth muscle and elastic fibers of the renal pelvis of Wistar rats, comparing control and penicillin G-treated animals. The animals were divided into 4 groups, normal rats (N), Wistar rats treated with penicillin G (NP); rats induced diabetes (D), diabetic Wistar rats with penicillin G (DP). The diabetes was induced in groups D and DP by alloxan. The fibrotic region of the renal pelvis was collected and reduced into small fragments. The sections were used for the transmission electron microscopy and stained by the following methods for optic microscopic: Iron Hematoxylin for disclosure of elastic fibers; Resorcin fuchsin for disclosure of elastic and elauninic fibers; Resorcin fuchsin after oxidation with 1% aqueous solution of oxone for disclosure of elastic, elauninic and oxytalan fibers; Azan evidencing the collagen and smooth muscle components; Picrosirius for observation of the collagen component (specifically type I and III); and Hematoxylin and Eosin, to show the cellular component. Microscopic and histomorphometry analysis showed that penicillin G alters the fibrous components of the renal pelvis, increasing areas of smooth muscle fibers and collagen type III deposition and decreasing mature elastic fibers (in this case, only between N and NP). Diabetes mellitus proved to be a metabolic disease also able to alter the morphology of the pelvis, leading to the augmentation of smooth muscle fiber area. Moreover, the area of type I collagen and the amount of mature elastic and elauninic fibers were diminished, while oxytalan fibers increased, together with a remarkable increase in the number of mitochondria. We can infer that the antibiotic therapy made by penicillin G and the diabetes, cause structural and ultrastructural differences in the renal pelvis of rats, mainly in the organization of elastic fiber, muscular and collagen components.

Chez des patients allergiques, il est possible de retrouver dans leur sérum desanticorps spécifiques du médicament (IgE) et dans leur sang des lymphocytes T spécifiquesdu médicament. La présence de LT spécifiques du médicament chez des patients allergiquessuggère la présentation du médicament par des cellules présentatrices d’antigène telles queles cellules dendritiques.Nous nous sommes alors intéressés à mieux comprendre l’implication deslymphocytes T et des cellules dendritiques dans le développement des allergies auxantibiotiques comme la pénicilline G (ou Benzyl-Pénicilline) ou le sulfaméthoxazole.Ce travail de thèse a permis: i) de démontrer la présence de lymphocytes Tspécifiques de la pénicilline G dans le sang périphérique de donneurs non allergiques à unefréquence mesurable, ii) de développer deux approches expérimentales et de modélisationpour l’identification des épitopes potentiellement présentés aux lymphocytes T et iii)d’étudier l’effet des médicaments sur les cellules dendritiques.Les perspectives de ce travail sont de mieux comprendre les mécanismes impliquésdans les allergies médicamenteuses au niveau des lymphocytes T et des cellules dendritiqueset de développer des tests de prédiction du « potentiel allergique » des médicaments, afinde mieux prédire les allergies médicamenteuses lors du développement des médicaments. / In allergic patients, antibodies like IgE or T-lymphocytes specific for the drugimplicated can be detected and measured. Presence of T-lymphocytes specific for the drugin allergic patients suggested the presentation of the culprit drug to T-lymphocytes byantigen presenting cells like dendritic cells.We were interested in better understanding the implication of T-lymphocytes anddendritic cells in the development of antibiotics allergies such as penicillin G (or Benzyl-Penicillin) or sulfamethoxazole.The results obtained in this work allowed us: i) to demonstrate the presence ofbenzyl-penicillin-specific T cells in the peripheral blood of non-allergic donors, at adetectable frequency; ii) to develop two approaches: one experimental and one usingmodelisation for the identification of epitopes potentially presented to T-lymphocytes andiii) to study the effect of drugs on DC.The perspectives of this research work are to better understand the mechanismsimplicated in drug allergies and to develop predictive tests for “drug allergic potential", inorder to be able to better predict drug allergies during drug development.

<p>Toxic products, such as endotoxin from the gram-negative and exotoxin from the gram-positive bacteria, are the most important initiators of the inflammatory host response in sepsis. In addition to antibacterial treatment, numerous attempts have been made to interfere with the exaggerated proinflammatory cascade initiated by the toxins. As most antitoxic and anti-inflammatory agents have shown no clear efficacy, an attractive alternative has been to prevent or minimise their release. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific β-lactam antibiotics.</p><p>There were significant reductions in endotoxin release from gram-negative bacteria when the combination of the PBP 3-specific β-lactam antibiotic, cefuroxime, and the protein synthesis inhibitor, tobramycin, was compared with cefuroxime alone. Increasing doses of tobramycin reduced endotoxin release and increased the killing rate. In a kinetic <i>in vitro</i> model the endotoxin release from <i>E.coli</i> was higher after the second dose of cefuroxime. Nevertheless, it was reduced after addition of tobramycin.</p><p>No binding of tobramycin to endotoxin was observed, either <i>in vivo</i> or <i>in vitro</i>. In a porcine sepsis model, a possible anti-inflammatory effect of ceftazidime and tobramycin, expressed as late cytokine inhibition, was seen.</p><p>The protein synthesis inhibitor, clindamycin, released less streptococcal pyrogenic exotoxin A (SpeA) from a group A streptococcus strain than penicillin, and addition of clindamycin to penicillin resulted in less toxin production than penicillin alone. The SpeA production was dependent on the bacterial number at the start of treatment. Higher doses of penicillin also led to less SpeA. </p><p>The choice of antibiotic class and dose may be important in the severely ill septic patient in whom an additional toxin release could be deleterious. A combination of a β-lactam antibiotic and a protein synthesis inhibitor seems beneficial but further investigations are needed.</p>

Toxic products, such as endotoxin from the gram-negative and exotoxin from the gram-positive bacteria, are the most important initiators of the inflammatory host response in sepsis. In addition to antibacterial treatment, numerous attempts have been made to interfere with the exaggerated proinflammatory cascade initiated by the toxins. As most antitoxic and anti-inflammatory agents have shown no clear efficacy, an attractive alternative has been to prevent or minimise their release. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific β-lactam antibiotics. There were significant reductions in endotoxin release from gram-negative bacteria when the combination of the PBP 3-specific β-lactam antibiotic, cefuroxime, and the protein synthesis inhibitor, tobramycin, was compared with cefuroxime alone. Increasing doses of tobramycin reduced endotoxin release and increased the killing rate. In a kinetic in vitro model the endotoxin release from E.coli was higher after the second dose of cefuroxime. Nevertheless, it was reduced after addition of tobramycin. No binding of tobramycin to endotoxin was observed, either in vivo or in vitro. In a porcine sepsis model, a possible anti-inflammatory effect of ceftazidime and tobramycin, expressed as late cytokine inhibition, was seen. The protein synthesis inhibitor, clindamycin, released less streptococcal pyrogenic exotoxin A (SpeA) from a group A streptococcus strain than penicillin, and addition of clindamycin to penicillin resulted in less toxin production than penicillin alone. The SpeA production was dependent on the bacterial number at the start of treatment. Higher doses of penicillin also led to less SpeA. The choice of antibiotic class and dose may be important in the severely ill septic patient in whom an additional toxin release could be deleterious. A combination of a β-lactam antibiotic and a protein synthesis inhibitor seems beneficial but further investigations are needed.

Protein engineering can be performed by combinatorial techniques (directed evolution) and data-driven methods using machine-learning algorithms. The main characteristic of directed evolution (DE) is the application of an effective and efficient screen or selection on a diverse mutant library. As it is important to have a diverse mutant library for the success of DE, we compared the performance of DNA-shuffling and recombination PCR on fluorescent proteins using sequence information as well as statistical methods. We found that the diversity of the libraries DNA-shuffling and recombination PCR generates were dependent on type of skew primers used and sensitive to nucleotide identity levels between genes. DNA-shuffling and recombination PCR produced libraries with different crossover tendencies, suggesting that the two protocols could be used in combination to produce better libraries. Data-driven protein engineering uses sequence, structure and function data along with analyzed empirical activity information to guide library design. Boolean Learning Support Vector Machines (BLSVM) to identify interacting residues in fluorescent proteins and the gene templates were modified to preserve interactions post recombination. By site-directed mutagenesis, recombination and expression experiments, we validated that BLSVM can be used to identify interacting residues and increase the fraction of active proteins in the library. As an extension to the above experiments, DE was applied on monomeric Red Fluorescent Proteins to improve its spectral characteristics and structure-guided protein engineering was performed on penicillin G acylase (PGA), an industrially relevant catalyst, to change its substrate specificity.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

The objective of this study is to identify and describe the cases reported / investigated of Congenital Syphilis in the Santa Maria’s University Hospital (HUSM), Rio Grande do Sul, Brazil, in the period of 2010 to 2014. Is a retrospective, cross-sectional, study in which secondary data found in records of notification/investigation of Congenital Syphilis Information System notification where evaluated. 128 records notification/investigation Congenital Syphilis where evaluated. The survey instrument was developed based on research of Congenital Syphilis records and pregnant women, where with the data collected has built up a database, which was submitted to descriptive statistical analysis. The analyzed variables consisted on the genitor: sociodemographic; realization or not of the prenatal care; period of maternal diagnosis of the Syphilis, nontreponemal test achievement in prenatal and childbirth; Prenatal treponemic test and delivery; treatment performed in pregnancy, partner treatment. The variables relating to infant were: not treponemal test, cerebrospinal fluid change, not treponemic test cerebrospinal fluid; radiological diagnosis; clinical diagnosis; in child treatment regimen; developments. Results: 128 cases of congenital syphilis were reported during the study. Pregnant women analyzed were the age from 20 to 35 years old, all of them of white color, educational level of 5th to 8th grade incomplete, underwent the prenatal care, had the diagnosis carried out in the third-trimestrer or childbirth, with inadequate treatment and with partners who were not treated. Newborns were male, white, have been notified within seven days after the birth date, treponemal test in peripheral blood reagent and asymptomatic in most cases. Three cases with non treponemal test in the reagent liquor six cerebrospinal change and a case with abnormal X-ray of the long bones, and more than half of the cases received treatment with Penicillin Cristalina for 10 days. Conclusion: Persisting vertical transmission, there is failure in the health care network focused on prenatal quality. / O objetivo deste estudo é identificar e descrever os casos notificados/investigados de sífilis congênita no Hospital Universitário de Santa Maria (HUSM) no Estado do Rio Grande do Sul-Brasil, no período de 2010 a 2014. Constitui um estudo retrospectivo, transversal, no qual avaliou dados secundários encontrados nas fichas notificação/investigação do Sistema Informação de Agravos de Notificação (SINAN) de sífilis congênita. Foram avaliadas 128 fichas de notificação/investigação de Sífilis Congênita. O instrumento de pesquisa foi elaborado com base nas fichas de investigação de sífilis congênita e em gestantes, os dados coletados construiu-se um banco de dados o qual foi submetido à análise estatística descritiva. As variáveis analisadas consistiram relativas a genitora: sociodemografica; realização do pré-natal; período do diagnóstico materno, realização de teste não treponêmico no pré-natal e no parto; teste treponêmico pré-natal e parto; tratamento realizado na gestante, tratamento do parceiro. As variáveis referentes à criança são idade, cor, sexo teste não treponêmico, alteração liquórica, teste não treponêmico no liquor; diagnóstico radiológico da criança; diagnóstico clínico; esquema de tratamento na criança; evolução do caso. Como resultados: foram notificados 128 casos de sífilis congênita no período de estudo. As gestantes analisadas tinham faixa etária de 20 a 35 anos, da cor branca, nível de escolaridade de 5º a 8º serie incompleta, realizaram o pré-natal, diagnóstico realizado no 3º trimestre ou no parto, com tratamento inadequado e com parceiros que não foram tratados. Os recém-nascidos eram do sexo masculino, brancos, foram notificados em até sete dias da data de nascimento, teste treponêmico no sangue periférico reagente e assintomáticos na maior parte dos casos. Três casos com exame não treponêmico no liquor reagente, seis com alteração liquórica e um caso com alteração no Raio X dos ossos longos, sendo que mais da metade dos casos recebeu tratamento com penicilina cristalina por 10 dias. Com isso, conclui-se que persistindo a transmissão vertical, verificou-se falhas na rede de atenção à saúde voltada para um pré-natal de qualidade.

Streptococcus pneumoniae, le pneumocoque, est une bactérie pathogène qui entraîne le décès de plus d'un million et demi de personnes dans le monde chaque année. Les β-lactamines sont très utilisées pour traiter les infections à pneumocoques. Ces antibiotiques inhibent la synthèse du peptidoglycane, une molécule géante constituant un réseau de chaînes glycopeptidiques qui englobe la cellule, lui confère sa forme et lui permet de maintenir son intégrité face à la pression osmotique. Le mécanisme d'action des β-lactamines est bien connud'un point de vue biochimique. En revanche, la réponse physiologique empêchant la multiplication des bactéries traitées est mal connue. Au cours de ma thèse, j'ai étudié les mécanismes moléculaires de la morphogenèse du pneumocoque par des approches de biochimie et de microbiologie. Un modèle de morphogenèse est proposé intégrant mes résultats à la littérature et permettant de formuler des hypothèses sur la réponse physiologique de S. pneumoniae aux β-lactamines. / Streptococcus pneumoniae, the pneumococcus, is a bacterial pathogen that causes more than 1.5 million deaths each year in the world. β-Lactams are widely used to treat patients with pneumococcal infections. These antibiotics inhibit the synthesis of the peptidoglycan, a giant molecule constituting a mesh of aminosugar strands encasing the cell. This main constituent of the cell wall allows cells to maintain their integrity under the turgor pressure, and endows bacteria with their shape. The action of β-lactams is well understood from a biochemical point of view. However, a complete understanding of the physiological response of treated bacteria remains elusive. In this thesis, I investigated the molecular mechanisms of the morphogenesis of S. pneumoniae using methods of biochemistry and microbiology. A morphogenesis model is built based on my results and the literature, which permits to emit hypotheses concerning the response of the pneumococcus to β-lactams.