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No description available.

Campylobacter jejuni (C. jejuni) is a food-borne bacterial pathogen that is the leading cause of Traveller’s Diaharea and Guillian-Barré Syndrome. Previous research determined that bacterial surface carbohydrates are virulence factors. Specifically, the study of the capsular polysaccharide (CPS) structures has widespread applications to understanding serotype cross-reactivity and biosynthetic pathways, the function of bacterial surface carbohydrates, and glycoconjugate vaccine development. This thesis characterized the CPS of C. jejuni HS:10 and subsequently used the CPS for developing a glycoconjugate vaccine. This was accomplished through extraction and purification of sugar from the bacteria, followed by characterization using chemical degradation, GC-MS, NMR and conjugation techniques. These analyses determined that the CPS was composed of a 3-linked GalNAc backbone and 6d-Galhepf attached at the 4th position with non-stoichiometrically attached O-methyl phosphoramidate attached to the 3 position of the heptose. Using this structure, a successful glycoconjugate vaccine was prepared using periodate oxidation and reductive amination.

Campylobacter jejuni bacterial pathogen is among the primary causes of food-borne acute gastroenteritis in North America and the world. It has also been linked to severe post-infection sequelae such as Guillain-Barré syndrome. Previous studies identified C. jejuni surface capsular polysaccharide (CPS) as a target for creation of a carbohydrate based vaccine in which the CPS is conjugated to a carrier protein. In this thesis, following sample purification, aspects of C. jejuni HS:5 CPS structure were characterized using numerous analytical techniques such as NMR and GC-MS. CPS is comprised of α-DD-Heptoses linked at C2 to the anomeric carbons of glucose. The α-Glucose molecules are linked though C4 to the α-DD-Heptose anomeric carbon. The α-DD-Heptose structure also has an occasional ring structured amino acid modification. Following characterization the CPS was oxidized and developed into a prototype glycoconjugate vaccine using TEMPO oxidation and EDC-CRM197 coupling methods. / The Natural Sciences and Engineering Research Council of Canada (NSERC)

Staphylococcus aureus is the leading cause of nosocomial infections in the US and is becoming increasingly difficult to treat due to the limited antibiotics available. Capsular polysaccharides (CP), a virulence factor produced by the bacterium, allows S. aureus to evade the uptake and killing by host neutrophils. It has been shown previously that CP serotype 5 retains more cell-associated CP while type 8 tends to release more CP into the supernatant. This research focused on whether this phenomenon is dependent upon the serotype-specific capHIJK genes that vary between the two serotypes. 6850, a methicillin- sensitive S. aureus (MSSA) serotype 8 strain, is a well characterized clinical isolate that was used in this study. This strain was subjected to two allelic replacement steps: the first step to replace the cap8HIJK genes with an ermB cassette, creating mutant 6850 (CP-); the second step to replace the ermB cassette with the cap5HIJK genes, which resulted in the creation of mutant 6850 (CP5). All 3 strains were characterized genotypically by PCR and phenotypically for growth rate, metabolic profile, and CP production. ELISA inhibition studies revealed that serotype 5 and the serotype 8 variants of S. aureus 6850 produced similar levels of cell-associated CP. These results suggest that cell wall anchoring of S. aureus CP5 and CP8 is not serotype specific, but instead is dependent on the genetic background of the bacterial strain. A better understanding of the anchoring mechanism may allow for development of alternative immunotherapeutics for S. aureus.

MESQUITA, Charles Jean Gomes de. Dimensão fractal e histometria digital na avaliação dos efeitos do propranolol sobre a reação capsular ao implante de silicone. 2014. 50 f. Tese (Doutorado em Cirurgia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2014. / Submitted by denise santos (denise.santos@ufc.br) on 2014-12-08T13:50:22Z No. of bitstreams: 1 2014_tese_cjgmesquita.pdf: 409219 bytes, checksum: faeed2fca385c9d082980d0e7e5e4eae (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2014-12-08T13:51:21Z (GMT) No. of bitstreams: 1 2014_tese_cjgmesquita.pdf: 409219 bytes, checksum: faeed2fca385c9d082980d0e7e5e4eae (MD5) / Made available in DSpace on 2014-12-08T13:51:21Z (GMT). No. of bitstreams: 1 2014_tese_cjgmesquita.pdf: 409219 bytes, checksum: faeed2fca385c9d082980d0e7e5e4eae (MD5) Previous issue date: 2014 / Despite their popular use in breast augmentation and reconstruction surgeries, the limited biocompatibility of silicone implants can induce severe side effects, including adverse capsularcontracture (ACC), an excessive foreign body reaction that forms, by unknown reason, a tight and hard fibrous capsule around the implant. A non-surgical treatment for ACC will be desirable as revision surgery for capsulotomy or capsulectomy with implant exchange are associated with a high risk of recurrence and complications. Recently it has been shown that sympathetic denervation accelerates wound contraction and delays reepithelialization in rats. Another study demonstrated that beta-adrenoceptor blockade has antifibrotic effects in a murine model of nonsinusoidal liver fibrosis. This study examines the effects of using propranolol, a non-selective beta-adrenoceptor antangonist, to prevent capsular formation around texturized silicone prosthesis implanted in the dorsum of male guinea-pigs. Animals (n=36) were randomly distributed into two equal groups, untreated or orally treated with propranololol (10 mg/kg dissolved in daily water). Capsules and implants were removed and examined for inflammation, thickness, fibrosis progression, density of type I and III collagen and fractal dimension by histological scoring using hematoxilin-eosin stained samples, digital histometry for measuring capsular thickness, picrosirius-polarization and digital image analysis for type I and type III collagen density and fractal dimension calculation by box-counting after 7, 14 or 21 days.Propranolol significantly reduced inflammation scores in the capsular tissue as compared to untreated group. Also, the capsular content of type I and type III collagen showed a statistical difference among the groups at different time points (p< 0,0001, Kruskal-Wallis test). The type I collagen density observed at 1, 2 or 3 weeks decreased significantly compared with that found in the control group. Conversely, the density of type III collagen increased in propranolol-treated group along the time.The capsule thickness in propranolol-treated cavies was significantly thinner and exhibited higher collagen type III/type I ratios and more irregular collagen fiber alignments than control animals. The observed decrease in fractal dimension of collagen also supported the alleviation of capsular formation by propranolol usage. Taken together, these data show that propranolol efficiently delays formation (antiproliferative or citotoxity effects) and maturation (antifibrogenic effect) of capsule around textured implants. Therefore, the blockade of beta-adrenoceptors is a promising option to support future therapeutic strategies in the treatment/prevention of capsular contracture. / As mamoplastias com implante de silicone são as cirurgias plásticas mais realizadas no mundo. O organismo reage a qualquer implante, encapsulando-o. Por razões desconhecidas, algumas cápsulas tornam-se patologicamente ativas gerando uma contratura capsular adversa (CCA), fibrose constritiva que deforma a mama reduzindo o resultado estético. Esta afecção de difícil tratamento requer, em alguns casos, reintervenções cirúrgicas que não impedem a recidiva. Tratamentos adjuvantes com drogas e/ou métodos físicos apresentam respostas variáveis e frequentemente insatisfatórias. O propranolol, antagonista não seletivo de betadrenoceptores, vem sendo empregado em estudos pré-clínicos em que a resposta fibrótica é importante parâmetro de avaliação e prognóstico. A demanda por métodos quantitativos e a tradução dos dados de bancada à clínica é uma constante. Métodos tradicionais como a histo-morfometria e a picrosirius-polarização tem sua acurácia aumentada quando associados a técnicas computacionais. A análise da Dimensão Fractal (DF), metodologia não linear de mensuração, tem se mostrado útil na avaliação de imagens biológicas permitindo visualização e compreensão de detalhes imperceptíveis ao olhar humano. O objetivo desse trabalho foi associar essas tecnologias (ferramentas computacionais e recursos matemáticos) na avaliação quantitativa dos efeitos do propranolol na formação da cápsula fibrosa peri-implante. Foram implantadas no plano subcutâneo 36 minipróteses texturizadas, preenchidas com gel de silicone (20 mL), no dorso de cobaias (Cavia porcellus). Os animais foram divididos aleatoriamente em dois grupos (n=18, cada) sendo tratados ou não com propranolol (10 mg/Kg dissolvido na água de beber diária (150 mL)). Os animais foram acompanhados e sacrificados após 7, 14 ou 21 dias. Após a retirada em bloco e explantação cuidadosa da prótese, a cápsula e o tecido do peri-implante foram preparados e corados pela hematoxilina-eosina (HE) ou picrosirius-red (PSR). A inflamação foi quantificada por atribuição de escores. Microfotografias digitais das amostras coradas pelo PSR foram obtidas sob polarização de forma padronizada para mensurar a espessura da cápsula, densidade do colágeno tipo I e tipo III, índice de fibrose (densidade do colágeno/espessura capsular) e cálculo da dimensão fractal pelo método de contagem de caixas. Foi utilizada análise estatística paramétrica ou não paramétrica conforme a natureza dos dados, considerando um nível de significância P < 0,05. Não ocorreram casos de infecção, seroma, hematoma ou deslocamento do implante. Todos os animais, independentemente do grupo e do momento do sacrifício exibiam uma cápsula fibrosa. As cápsulas dos animais tratados com propranolol exibiam significantemente menor intensidade de inflamação, cápsulas menos espessas, menor índice fibrótico e menores valores de dimensão fractal do que as do grupo controle. A DF revelou correlação inversa com esses parâmetros. Em conjunto, estes achados são condizentes com a inibição da formação da cápsula pelo betabloqueio sistêmico não seletivo, sugerindo ação anti-inflamatória e antifibrogênica do Propranolol.

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CARACTERISATION DE VACCINS A BASE DE GLUCIDESVariables influençant l'immunogénicité et propriétés physico-chimiques des vaccins glycoconjuguésDe nombreux aspects peuvent influer sur l'immunogénicité des vaccins conjugués et les principales variables étudiées jusqu'ici sont la taille du fragment saccharide et la nature des glycosides: taux de protéine dans le conjugué purifié, la stratégie de conjugaison, nature de l’espaceur et la protéine porteuse.La taille de la partie saccharidique et le ratio de cette partie / protéine a été étudiée dans différents travaux de Seppälä et Mäkelä,.Dans l'une des premières études sur l'effet de la taille sur l'immunogénicité de la protéine conjuguées à des dextrans, il a été montré que des dextrans de faible poids moléculaire conjugué à l’albumine sérique de poluet induit des réponses anti-dextran fortes chez la souris. L’augmentation de la taille du dextran, a abouti à une réduction de l’immunogénicité. Peeters et al. a montré qu’un tétramère synthétique de Hib unité capsulaire polysaccharide, conjuguée à un support protéique, induit chez des souris adultes, des niveaux d'anticorps primates non humains comparables à un conjugué Hib commercial. Ces niveaux sont plus élevées que ceux induits par un trimère répété, ce qui indique que pour le Hib un minimum de huit sucres est nécessaire pour une bonne réponse immunologique. Laferrière et al. a trouvé peu d'influence de la longueur de la chaîne glucidique sur l'immunogénicité des vaccins antipneumococciques conjugués chez la souris. Pozsgay et al. a étudié chez la souris, l'immunogénicité de l’oligosaccharides du LPS de Shigella dysenteriae de type 1 conjugué à l'albumine sérique humaine (HSA). Les auteurs ont constaté que les octa-, dodéca-, et des fragments de hexadécasaccharides induit des niveaux élevés d'anticorps IgG après trois injections. Ces niveaux sont supérieurs à ceux obtenus avec un conjugué tétrasaccharidique. L'influence du ratio glucides / protéine est différente pour les trois conjugués. Le conjugué octasaccharide-HSA avec la plus forte densité provoque une bonne réponse immunitaire, tandis que dans le cas des conjugués dodéca- et hexadécasaccharides, la densité médiane est optimal. Ces études suggèrent que la longueur de la chaîne d'oligosaccharides et le chargement de l’haptène peuvent être liés entre eux pour déterminer l'immunogénicité des vaccins glycoconjugués.L'espaceur est une molécule linéaire courte qui est généralement liée à la chaîne polysaccharidique et la protéine ou de fragments. Il ya des évidences dans la littérature qui suggèrent que les espaceurs rigides, contraints comme le maléimide cyclohexyle, provoquent une importante quantité d'anticorps indésirables, avec le risque de conduire à une réponse immunitaire éloignée de l'épitope ciblé sur la haptène. L'utilisation d'un alkyle souple type maleimido a été rapporté comme un moyen de surmonter l'immunogénicité observée précédemment. Un certain nombre de transporteurs protéiques ont été utilisés jusqu'ici dans l'évaluation préclinique et clinique de vaccins conjugués. Des protéines telles que les anatoxines diphtériques et tétaniques, qui dérivent des toxines respectives, après la décontamination chimique avec le formaldéhyde, ont été initialement choisies comme transporteur en raison de inocuité (tétanos et la vaccination contre la diphtérie). CRM 197, un mutant non toxique de la toxine 61 de la diphtérie a été largement utilisé comme support pour Hib. Un complexe protéique de la membrane externe de méningocoque du sérogroupe B a été utilisé par Merck comme support pour leur vaccin conjugué Hib. GSK dans leur vaccin antipneumococcique, conjugué multivalent, introduit la protéine D Hib liée à la plupart des polysaccharides inclus dans le vaccin. L'équipe de John Robbins fait un large usage de la forme recombinante non toxique de l’exo-toxine de Pseudomonas aeruginosa comme support contre Staphylococcus aureus de type 5 et 8 ainsi que pour Salmonella. / CHARACTERIZATION OF CARBOHYDRATE BASED VACCINES Variables influencing the immunogenicity and physicochemical properties of glycoconjugate vaccinesMany aspects can influence the immunogenicity of conjugate vaccines and the main variables investigated so far are the size of the saccharide moiety, the saccharide:protein ratio in the purified conjugate, the conjugation strategy, the nature of the spacer and the protein carrier. The size of the saccharide moiety and saccharide/protein ratio were investigated in different works such as Seppälä and Mäkelä in one of the first studies on the effect of size and chemistry on the immunogenicity of dextrans-protein conjugates found that dextrans of low molecular weight conjugated to chicken serum albumin, induced strong anti-dextran responses in mice, while increasing the dextrans' size resulted in reduced immunogenicity.47 Peeters et al. showed that a synthetic tetramer of Hib capsular polysaccharide repeating unit, conjugated to a protein carrier, induced in adult mice and non-human primates antibody levels comparable to a commercial Hib conjugate and higher than those induced by a trimer, indicating that for Hib a minimum of eight sugars is needed for a proper immunological response.48 Laferriere et al. found little influence of the carbohydrate chain length on the immunogenicity of pneumococcal conjugate vaccines in mice.49 Pozsgay et al. studied the immunogenicity in mice of synthetic Shigella dysenteriae type 1 LPS oligosaccharides conjugated to human serum albumin (HSA). The authors found that octa-, dodeca-, and hexadecasaccharide fragments induced high levels of lipopolysaccharide binding IgG antibodies in mice after three injections and were superior to a tetrasaccharide conjugate. The influence of the carbohydrate/protein ratio was different for the three conjugates. The octasaccharide-HSA conjugate with the highest density evoked a good immune response, while in the case of dodeca- and hexadecasaccharide conjugates, the median density was optimal.50 These studies suggest that oligosaccharide chain length and hapten loading might be interconnected in determining the immunogenicity of glycoconjugate vaccines. The spacer is a short linear molecule that is generally linked to the polysaccharide chain or to the protein or to both moieties, depending on the chemistry, used to facilitate the coupling between the protein and sugar. There are evidences in the literature which suggest that rigid, constrained spacers like cyclohexyl maleimide, elicit a significant amount of undesirable antibodies, with the risk of driving the immune response away from the targeted epitope on the hapten.51 52 The use of a flexible alkyl type maleimido spacer has been reported as a way to overcome the previous observed immunogenicity of cyclic maleimide linkers.53 A number of protein carriers have been used so far in preclinical and clinical evaluation of conjugate vaccines. 54 55 56 57 58 59 60Proteins such as diphtheria and tetanus toxoids, which derive from the respective toxins after chemical detoxification with formaldehyde, were initially selected as carrier because of the safety track record accumulated with tetanus and diphtheria vaccination. CRM197, a non-toxic mutant of diphtheria toxin61 which instead does not need chemical detoxification, has been extensively used as carrier for licensed Hib, pneumococcal, meningococcal conjugate vaccines and for other vaccines being developed. An outer membrane protein complex of serogroup B meningococcus has been used by Merck as carrier for their Hib conjugate vaccine.62 GSK in their multivalent pneumococcal conjugate vaccine introduced the use of the Hib-related protein D as carrier for most of the polysaccharides included into the vaccine.63 64 The team of John Robbins made extensive use of the recombinant non toxic form of Pseudomonas aeruginosa exo-toxin as carrier for Staphylococcus aureus type 5 and 8 as well as for Salmonella

Streptococcus pneumoniae (pneumococo) é um importante patógeno humano, responsável por graves infecções das vias respiratórias. O principal fator de virulência desse microrganismo é a cápsula polissacarídica (PS), antígeno das vacinas atuais, que são elaboradas com os PS purificados de cepas de pneumococo prevalentes na população. O objetivo desse trabalho foi desenvolver um processo de purificação do PS do sorotipo 14, responsável por 39% das doenças em crianças de 0-6 anos no Brasil. A metodologia de purificação envolveu separação celular por microfiltração tangencial e concentração do microfiltrado com membrana de ultrafiltração tangencial de 50 kDa. O produto dessa etapa foi diafiltrado com dodecil sulfato de sódio em membrana de ultrafiltração tangencial de 30 kDa, seguido de precipitação com ácido tricloroacético a 5%, precipitação por etanol (20% e 60%) e cromatografia de troca aniônica. A pureza do PS foi avaliada pelo conteúdo de proteínas e ácidos nucleicos remanescentes e o tamanho por cromatografia de exclusão molecular. O PS foi obtido com pureza e tamanho requeridos pelos órgãos regulatórios e o rendimento final do processo foi de 65%. / Streptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for severe respiratory tract infections. The main virulence factor of this microorganism is the capsular polysaccharide (PS), which is the antigen of all current vaccines, which are prepared with purified PS of pneumococcal strains prevalent in the population. The objective of this work was to develop a new purification process for PS of serotype 14, responsible for 39% of diseases in children of 0-6 years old in Brazil. The purification method involved cell separation by tangential microfiltration and concentration of cell-free culture broth containing PS by tangential ultrafiltration (50 kDa). The product of this step was diafiltrated with sodium dodecyl sulfate by tangential ultrafiltration (30 kDa), following by 5% trichloroacetic acid precipitation, 20% and 60% ethanol precipitation and anion exchange chromatography. The PS purity was evaluated by the content of residual proteins and nucleic acids, and the molecular mass by size exclusion chromatography. The purity and molecular mass requirements were achieved and the process global yield was 65%.