Abstract The aims of this work were to study the carriage of respiratory bacteria and to identify risk factors affecting pharyngeal colonisation by these pathogens among young Finnish men during military service, and also to investigate the role of mannose-binding lectin (MBL) concentrations and MBL2 gene polymorphisms in the carriage of respiratory bacteria. A total of 892 military recruits entering the Kainuu Brigade, including 224 men with asthma, were followed up prospectively to the end of their military service. Carriage of Streptococcus pneumoniae, Neisseria meningitidis and beta-haemolytic streptococci appeared to be higher during and at the end of military service than at the beginning. Smoking was found to be a significant risk factor for colonisation by these bacteria. S.pneumoniae was more common in the asthmatic than military conscripts in the non-asthmatic ones at the beginning of military service. A low MBL level increased the risk of carrying N. meningitidis and beta-haemolytic streptococci during military service among non-smokers but not among smokers. Low MBL levels producing MBL2 haplotypes seemed to be associated with the carriage of N. meningitidis and S. pneumoniae. Characterisation of all the oropharyngeal N.meningitidis isolates obtained (n=215) by phenotypic and genotypic methods showed that most of them belonged to the carriage-associated ST-60 clonal complex. Clonal complexes ST-41/44, ST-32, and ST-23, which have previously been associated with disease, also accounted for a third of the carriage strains. Furthermore, a significant association was indicated between an acute upper respiratory infection and oropharyngeal carriage of the virulent meningococcal ST-23 clone. In conclusion, the results reported here show a significant increase in bacterial carriage during military service and provide new information on the association between MBL and carriage of respiratory bacteria. These findings also highlight the importance of smoking cessation, especially among military conscripts, who have been found to be a risk group for invasive bacterial diseases, and they also point to the importance of meningococcal vaccination for military recruits and the need for an efficacious vaccine against serogroup B meningococci. / Tiivistelmä Hengitystieinfektiot ovat yleisiä varusmiespalvelun aikana. Myös oireeton bakteerien nielukantajuus on lisääntynyt. Useimmiten infektiot ovat lieviä virusinfektioita, mutta bakteerien nielukantajuus voi johtaa myös vaikeisiin bakteeritulehduksiin. Tämän väitöskirjatyön tarkoituksena oli tutkia bakteerien nielukantajuutta varusmiespalveluksen alkaessa ja päättyessä sekä mahdollisten hengitystieinfektioiden aikana ja näin saada uutta tietoa bakteerien nielukantajuuteen vaikuttavista tekijöistä. Lisäksi tavoitteena oli selvittää mannoosia sitovan lektiinin (MBL) sekä MBL2-geenin polymorfismien yhteyttä bakteerien nielukantajuuteen. Työn tarkoituksena oli myös feno- ja genotyypittää varusmiehiltä palveluksen aikana eristetyt meningokokkikannat ja verrata niitä vastaavana ajankohtana invasiivista tautia sairastaneista henkilöistä eristettyihin meningokokkikantoihin. Tutkimuksessa seurattiin prospektiivisesti 892 varusmiestä, jotka suorittivat asepalveluksen Kainuun Prikaatissa vuosina 2004–2006. Tutkimukseen osallistuneista varusmiehistä 224:llä oli astma. Tutkimuksessa havaittiin, että oireeton bakteerien nielukantajuus lisääntyy merkitsevästi varusmiespalveluksen aikana. Lisäksi havaittiin, että tupakointi oli merkittävä itsenäinen riskitekijä pneumokokin, meningokokin sekä beta-hemolyyttisten streptokokkien nielukantajuudelle varusmiespalveluksen aikana. Astmaatikkojen pneumokokin nielukantajuus varusmiespalveluksen alussa oli yleisempi kuin terveiden varusmiesten. Tutkimuksessa osoitettiin myös pienen seerumin MBL-pitoisuuden sekä MBL2-geenin polymorfismin eksoni 1:n alueella ja geenin säätelyalueella olevan riskitekijöitä meningokokin, pneumokokin sekä beta-hemolyyttisten streptokokkien nielukantajuudelle tupakoimattomilla varusmiehillä. Meningokokin nielukannoista jopa kolmasosa kuului genotyyppiryhmään, jonka on aiemmissa tutkimuksissa havaittu liittyvän invasiiviseen tautiin. Tutkimuksessa osoitettiin myös tietyn hyperinvasiivisen meningokokin genotyypin (ST-23) liittyvän hengitystieinfektioepisodeihin. Tässä väitöskirjatyössä osoitettiin, että bakteerien nielukantajuus lisääntyy merkitsevästi varusmiespalveluksen aikana ja että oireettomilla varusmiehillä tavataan myös hyperinvasiivisia meningokokkikantoja. Tutkimus antoi myös uutta tietoa hyperinvasiivisten meningokokin genotyyppien liittymisestä hengitystieinfektioihin sekä MBL:n vaikutuksesta bakteerien nielukantajuuteen. Tutkimushavainnot tukevat tupakoimattomuuden edistämisen tärkeyttä myös varusmiespalveluksen aikana.

Cystic fibrosis (CF) is a single gene Mendelian disorder characterized by pulmonary disease and pancreatic insufficiency. Pulmonary disease is the major cause of death in CF patients. Although some cystic fibrosis transmembrane conductance regulator (CFTR) genotypes are associated with less severe disease, patients possessing the same genotype show great variation in pulmonary disease severity and progression. Genes involved in modulating the inflammatory response and genes increasing susceptibility to infection are proposed as modifiers of pulmonary disease severity. Polymorphisms selected for based on evidence that they affect the function of the gene and prevalence of the putative risk allele: 1) antiprotease gene alpha-1-antitrypsin (alpha-1-AT), 2) innate immunity genes: mannose binding lectin (MBL2) (promoter [G→C] at -221 and codon 52 (Arg52Cys, D allele), 54 (Gly54Asp, B allele), and 57 (Gly57Glu, C allele), and pulmonary surfactant genes SPA-1 (Arg219Trp), SPA-2 (Thr9Asn, Lys223Gln) and SPD (Thr11Met), 3) antioxidant genes GSTM1 and T1 (gene deletion polymorphisms), GSTP1 (Ile105Val) and GCLC repeats, 4) mucin genes (MUC2 and MUC5B). Pulmonary disease progression and survival in patients with chronic Burkholderia cepacia complex (BCC) infection were also investigated controlling for genomovar and RAPD type of the organism. BCC infection was associated with more severe pulmonary disease progression and worse survival. Alpha-1-AT genotype was not a major contributor to variability of pulmonary disease severity, but the results suggest that alpha-1-AT plasma levels during pulmonary infections may be affected by poor nutritional status. We showed similar pulmonary disease progression and MBL2 genotype. Contrary to the previous literature, wild-type MBL2 genotype was associated with steeper decline in pulmonary disease over time following chronic infection with BCC, but genotype was not associated with increased susceptibility to BCC infection. We showed inconsistant results for the pulmonary surfactant gene polymorphisms, GSTM1, T1 and GSTP1 polymorphisms, and number of repeats for GCLC and MUC5B depending on the phenotype investigated. We conclude that some of the variability in pulmonary disease severity and progression in CF is explained by polymorphisms in secondary genes. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Virtually all extracellular proteins in humans are glycoproteins and likewise are many biopharmaceuticals. The glycosylation is directly correlated to biological function and stability of these proteins. The ability to isolate glycoproteins is thus of great importance in many applications. The most common isolation method for glycoproteins is affinity chromatography using lectins, a ubiquitous and versatile group of carbohydrate-binding proteins. The lectin Concanavalin A (ConA) has long been used for this purpose but suffers from undesired leakage into the eluate, causing an inquiry of alternative chromatography ligands or optimization of the ConA resin.In this study, a total of 20 different lectins, including ConA, were evaluated and compared in terms of suitability as ligands in affinity chromatography for glycoprotein isolation. The lectins’ binding to glycoproteins were studied, mainly through microtiter plate binding assays using a monoclonal IgG1 antibody and Conalbumin (Ovotransferrin). Further, sugar-specificities and potential eluting sugars for the lectins were examined through inhibition with eight different carbohydrates. Additionally, the glycoprotein binding and leakage of ConA columns were examined, and a potential leakagereducing treatment of ConA resin evaluated.ConA was found to be superior in binding to the investigated glycoproteins but exhibited a limited binding when immobilized to an agarose resin. This discrepancy is likely a consequence of structurally hidden glycans on the used glycoproteins and requirements of long residence time when used in a chromatographic setting. Binding competition with several sugars were investigated with a similar microtiter plate binding assay. This method displayed potential to predict the behaviour of sugars and their suitability as eluting agents in a chromatography column. The best eluting sugar for ConA was showed to be methylmannoside, ideally in combination with methylglucoside. Lastly, evaluation of ConA columns with a crosslinking glutaraldehyde-treatment showed that the ConA ligand leakage may be significantly reduced, although further studies and optimizations are needed.This study thus presents a repertoire of lectins and their differences in terms of glycoprotein-binding and sugar-specificity, as well as evaluations of ConA columns’ efficiency and potential leakage-prevention.

Les travaux rapportés dans ce mémoire concernent le développement de nouvelles réactions métallo-catalysées pour la création de liaison carbone-hétéroatome et carbone-carbone ainsi que leurs applications à la synthèse de produits biologiquement actifs. La première partie de ce manuscrit est consacrée à l'étude de la réactivité des sucres dans les couplages organométalliques. Des conditions ont été développées pour la création de la liaison C-S entre glycosyl thiols et partenaires arylés. De plus, la création de la liaison carbone azote de glycosyl amines avec des acides boroniques a été étudiée. Les produits synthétisés dans cette première partie ont été évalués pour leur potentiel d'inhibition de la Lectine A chez Pseudomonas aeruginosa, impliquée dans de sévères infections pulmonaires.La seconde partie de ce travail est dédiée à la création d'une série inédite d'analogues du 6BrCaQ, inhibiteurs de la Hsp90 ainsi que leur évaluation biologique. Cette nouvelle série est obtenue grâce à une nouvelle méthodologie de synthèse basée sur l'activation C-H entre un hétérocycle halogéné et son partenaire C-H activable. L'activité antiproliférative et l'inhibition de la Hsp90 ont été évaluées et seront présentées dans ce manuscrit. / The work reported in this dissertation concerns the development of new metal-catalyzed reactions for the creation of carbon-heteroatom and carbon-carbon bonds as well as their applications to the synthesis of biologically active products.The first part of this manuscript is devoted to the study of the reactivity of sugars as nucleophiles in organometallic couplings. Conditions were developed for the creation of the C-S bond between glycosyl thiols and aryl partners. Moreover, the creation of the nitrogen carbon bond of glycosyl amine with boronic acids was studied. The products synthesized in this first part have been evaluated for their potential to inhibit the lectin A, in Pseudomonas aeruginosa related lung infections.The second part of this work is dedicated to the creation of a new series of 6BrCaQ analogues as Hsp90 inhibitors and their biological evaluation. This new series was synthetized through a new CH activation methodology. The antitumoral potential was evaluated and will be presented in this manuscript.

Les lymphocytes T CD8+ jouent un rôle prépondérant dans le contrôle de l’infection par le VIH suggérant ainsi qu’un vaccin efficace doit induire une réponse cellulaire T CD8+ robuste et durable.En fournissant une source antigénique importante qui fait défaut chez les patients répondeurs aux poly-chimiothérapies antirétrovirales, la vaccination thérapeutique pourrait favoriser ce type de réponse cellulaire, non restaurée par les seuls traitements antirétroviraux. Les cellules dendritiques (DC) autologues, qu’elles soient chargées en virus inactivé, en peptides viraux ou en ARN messagers (ARNm) sont capables d’induire des réponses robustes des cellules T CD8+ contre les antigènes du VIH, faisant de bons candidats vaccins thérapeutiques.Dans ce projet, nous avons étudié l’efficacité d’une vaccination thérapeutique basée sur l’injection de DC autologues transfectées ex vivo avec des ARNm codant les protéines du VIH (projet initié en collaboration avec Guido Vanham, Institute of Tropical Medicine, Anvers). La composante antigénique du vaccin est fournie par les séquences virales endogènes du patient; ainsi nous espérons adapter la spécificité de la réponse immunitaire aux virus autologues. Nous travaillons avec le Macaque cynomolgus comme modèle animal d’infection par les virus d’immunodéficience (SIV), modèle pertinent dans l’approfondissement des connaissances en pathogénèse et en développement vaccinal, notamment pour tester l’innocuité et l’efficacité des vaccins basés sur l’utilisation des DC. La mise au point un système de culture de DC basé sur la prolifération puis la différenciation des progéniteurs hématopoïétiques CD34+, qui nous a permis de générer in vitro des DC matures en nombre suffisant pour la vaccination (au moins 10.106).Nous avons caractérisé ces DC phénotypiquement, avec une attention particulière vis-à-vis de l’expression de lectines de type C et d’autres récepteurs DC-spécifiques. Nous avons mis en évidence que l’état d’activation des DC définit le niveau d’expression de ces molécules. Ces récepteurs membranaires sont impliqués dans la capture et la présentation des antigènes aux effecteurs, ainsi que dans l’orientation de la réponse immune. Le phénotype des DC générées in vitro a été comparé au phénotype de plusieurs population de cellules présentatrices d’antigène présentes in vivo chez le macaque.La transfection avec des ARNm par électroporation est un moyen sûr et efficace de charger les DC en antigènes. Nous avons étudié dans un premier temps l’immunogénicité du vaccin chez des macaques sains. Les résultats montrent qu’une réponse cellulaire de type Th1 (IFN-gamma et interleukine-2) spécifique à Gag est induite dès la première injection et peut être renforcée après les injections suivantes. Nous avons également mis en évidence l’induction de lymphocytes T CD8+ capables de sécréter en réponse à Gag plusieurs de ces cytokines, IFNg, IL2, TNFa, la chimiokine MIP1b, ou encore le marqueur de dégranulation cytotoxique CD107a. En revanche, aucune sécrétion de cytokine de type 2 ni de réponse anticorps spécifique de Gag n'a pu être mise en évidence.La seconde partie de notre projet a consisté en l’étude d’une stratégie de vaccination par ciblage des DC in vivo avec des protéines de fusion DC-spécifiques associées à des antigènes. Ce travail a été mené en collaboration avec le Baylor Insitute for Immunology Research (BIIR) à Dallas. Nous avons tout d’abord sélectionné les meilleurs clones d’Ac spécifiques de molécules humaines pour leur réactivité croisée avec les molécules du macaque cynomolgus. L’immunogénicité de protéines de fusion (dirigées contre LOX-1 et DC-ASGPR couplées à Gag du VIH ou HA1 de Influenza), a ensuite été évaluée in vivo chez le macaque. Ces travaux ont confirmé in vivo que l’induction de différents profils de réponses immunitaires Ag-spécifiques (Th1 et IL10) est possible par ciblage in vivo des DC avec des protéines de fusion dirigées contre différentes molécules (LOX-1 et DC-ASGPR). / Current treatments against HIV infection would benefit from the development of complementary immunotherapeutic strategies. Dendritic cells (DC) play a major role in the induction of immune responses. In this thesis, we examine the use of the DC in therapeutic vaccination. Ex vivo loading of DC with messenger RNA encoding viral proteins and in vivo loading of DC by targeting antigen to DC-specific endocytic receptors are two strategies evaluated in healthy macaque.

Ce travail de thèse est consacré à la préparation de glycopolymères porteurs de groupements pendants mannoside d’heptyle et à l’évaluation de la capacité de ces ligands multivalents à inhiber la fixation bactérienne sur les cellules humaines. Nous avons synthétisé, par polymérisation radicalaire contrôlée, une série de glycopolymères linéaires ou en étoile présentant des masses molaires, des densités en mannoside et des microstructures modulables dans le but d’évaluer l’influence de ces paramètres sur les processus d’interactions avec diverses souches de bactéries E coli (AIEC LF82 et UTI 89). Nous avons tout d’abord mis en évidence par diffusion dynamique et statique de la lumière, la formation d’agrégats entre ces glycopolymères et FimH, la lectine à l’origine de la fixation de souches de bactéries E coli, traduisant des interactions fortes entre les motifs mannosides et les sites de reconnaissance au mannose de la lectine. Nous avons ensuite évalué l’aptitude de ces ligands multivalents à bloquer l’adhésion bactérienne d’AIEC LF82 (impliquée dans la maladie de Crohn) sur des cellules épithéliales intestinales T84. Il a été démontré en conditions in vitro que l’ajout de 10 nM ou 100 nM d’unités mannoside (respectivement en pré- ou post-incubation) réduit de moitié l’adhésion des bactéries sur les cellules épithéliales. L’effet anti-adhésif de ces glycopolymères a été confirmé par des tests ex vivo réalisés sur des intestins isolés de souris transgéniques CEABAC10. Enfin, nous avons exploité la technique de nanoprécipitation pour l’élaboration de nanocapsules de glycopolymères à cœur huileux. Le procédé développé permet la synthèse de nanocapsules de dimensions contrôlées, porteuses de groupements fonctionnels (fluorophores, ligands) ou de particules métalliques et l’encapsulation de molécules actives à cœur en une seule étape. / This PhD work focuses on the preparation of glycopolymers bearing pendent heptyl mannose groups and the evaluation of the capability of such multivalent ligands to inhibit bacterial adhesion to human cells. Aiming at understanding the impact of various structural parameters on glycopolymer/ E coli interactions (AIEC LF82 et UTI 89 strains of E. coli), a series of linear and star-shaped glycopolymers with tunable molecular weight, mannoside density and microstructure (block copolymers, gradient copolymers, random copolymers) has been constructed. The association of the glycopolymers with FimH adhesin, a lectin which possesses a mannose-specific receptor site and is responsible for recognition and binding to host cells, was first confirmed by static and dynamic light scattering experiments. The propensity of the glycopolymers to prevent attachment of E. coli (AIEC LF82 involved in Crohn’s disease) to intestinal epithelial cells (T84 cells) was further investigated through adhesion assays. It was shown that under in vitro conditions, the addition of 10 nM or 100 nM of glycopolymer on a mannose unit basis (in pre-incubation and post-incubation respectively) decreases by half the bacterial adhesion to intestinal epithelial cells. The anti-adhesive effect of these multivalent ligands was further confirmed in ex vivo conditions for colonic loops of transgenic CEABAC10 mice (Crohn’s disease model mouse). Finally we took advantage of the nanoprecipitation process to generate glyconanocapsules with oily core. The employed strategy allowed for preparing well-defined nanocapsules bearing groups of interest (tags, ligands) or metal particles within the shell and loaded with active molecules in the core in one step.

No description available.

<p>Synthetic carbohydrate carrying polymers have gained substantial attention recently due to their biocompatibility and their wide range of applications such as targeted drug delivery systems, gene therapy and cell-specific biomedical materials. The overall objective of this research is to develop novel carbohydrate bearing polymers through modification of polyvinylamine (PVAm) backbone with galactose groups and to discover potential applications for this new category of glycopolymers.</p> <p>PVAm-g-galactose (PVAm-GAL) with various molecular weights and grafting extents were prepared and characterized by nuclear magnetic resonance (NMR) and potentiometric and conductometric titration. Bonding of PVAm-GAL with phenylboronic acid modified PVAm (PVAm-PBA) and phenylboronate modified surfaces were studied on a quartz crystal microbalance with dissipation (QCM-D). Multilayer assembly of alternating layers of PVAm-GAL and PVA-PBA was formed on the silica sensor.</p> <p>Interaction of PVAm-GAL with RCA₁₂₀ lectin, a galactose specific protein, was studied on a silica sensor using QCM-D. Galactose binding proteins are overexpressed in hepatocyte and have been widely exploited for targeting the liver tissue with the help of galactosylated polymeric carriers. RCA₁₂₀lectin shows spontaneous adsorption on galactose rich surfaces obtained by the adsorption of PVAm-GAL on silica sensors. Association constant of the interaction was calculated . Effect of pretreatment with bovine serum albumin (BSA) was also examined.</p> <p>Cationic polymers can form polyelectrolyte complexes (PECs) with negatively charged DNA, resulting in formation of nano-sized complexes for gene delivery purposes. PECs based on PVAm-GAL and different DNA samples were prepared and their physicochemical properties were investigated using dynamic light scattering (DLS) and electrophoretic mobility measurements. Furthermore, PVAm-GAL was studied as coating for Ca-alginate beads which are widely used for cell encapsulation purposes. PVAm-GAL can strengthen the capsule’s surface and increase the physicochemical stability of the beads against chemical degradations. PVAm-GAL coated alginate beads successfully survived treatment with sodium citrate and high ionic strength solutions.</p> / Master of Applied Science (MASc)

Introdução: Chlamydia trachomatis (CT) é causa da infecção sexualmente transmitida de origem bacteriana mais comum. Na mulher, a infecção genital pela CT pode causar cervicite, uretrite, endometrite, salpingite. Infecções persistentes ou recorrentes provavelmente representam um importante fator de risco para o desenvolvimento de sequelas associadas, como dor pélvica crônica, gravidez ectópica e infertilidade por fator tubário. A lectina ligadora de manose (MBL), componente doo sistema imune inato, tem importante papel na defesa antimicrobiana, reconhecendo vírus, fungos e patógenos bacterianos. O gene que codifica para a MBL é polimórfico, e a substituição de um único nucleotídeo resulta na produção de uma proteína instável, que é rapidamente degradada. Objetivo: Avaliar se mulheres brasileiras portadoras de um polimorfismo do gene da MBL apresentam diferentes susceptibilidades para a ocorrência de obstrução tubária, na presença ou ausência de uma infecção prévia por Chlamydia trachomatis. Métodos: Em estudo caso-controle, foram avaliadas 75 pacientes com obstrução tubária e 75 pacientes com tubas pérvias, atendidas na Divisão de Ginecologia do Hospital das Clínicas da Universidade de São Paulo (HCFMUSP). Anticorpos IgG anti-CT foram mensurados através de um ensaio de imunoabsorção enzimática para investigar uma infecção prévia pela CT. Para o polimorfismo, realizou-se coleta de células bucais e o DNA extraído foi analisado através de reação em cadeia da polimerase (PCR), digestão de endonuclease e gel de eletroforese, utilizando pares de primers específicos para a região polimórfica. Todo material foi mantido a -80°C e enviado em gelo seco para a Division of Immunology and Infectious Diseases da Weill Cornell Medicine em Nova York. Associações entre genótipos de MBL ou alelos e permeabilidade tubária foram analisadas pelo teste de qui-quadrado de Pearson com ou sem correção de Yates. Resultados: Não houve diferença na detecção de anticorpos da CT entre os grupos. Mulheres com tubas obstruídas tiveram uma prevalência maior do genótipo AB (36%) versus (16%), resultado estatisticamente significativo (p < 0,01). De maneira semelhante, a distribuição do alelo A e do alelo variante B também apresentaram diferenças significantes entre os grupos (p < 0,01). Conclusão: Os achados sugerem, que embora a exposição à Chlamydia trachomatis tenha sido semelhante em ambos os grupos, a presença do alelo variante B do gene que codifica para a MBL aumenta o risco de desenvolvimento da obstrução tubária, subsequente à infecção pela CT ou outros agentes infecciosos. Nas mulheres brasileiras avaliadas a presença de tal polimorfismo genético aumentou a probabilidade de obstrução tubária em consequência de uma infecção do trato genital / Introduction: Chlamydia trachomatis (CT) is the cause of the most common bacterial sexually transmitted infection. In women, genital CT infection may cause cervicitis, urethritis, endometritis, salpingitis. Persistent or recurrent infections probably represent an important risk factor for the development of associated sequelae, such as chronic pelvic pain, ectopic pregnancy and tubal factor infertility. Mannose-binding lectin (MBL), a component of the innate immune system, has an important role in antimicrobial defense, recognizing viral, bacterial and fungal pathogens. The gene coding for MBL is polymorphic and a single nucleotide substitution results in production of an unstable protein, that is rapidly degraded. Objective: To evaluate whether Brazilian women with a polymorphism in the MBL gene present different susceptibilities to the occurrence of fallopian tube damage, in the presence or absence of a previous infection by CT. Method: In a case-control study, 75 patients with tubal obstruction and 75 patients with patent tubes were studied, all seen at the Gynecology Division of the Hospital das Clínicas of the University of São Paulo (HCFMUSP). IgG anti-CT antibodies were measured by enzyme-linked immunoassay to investigate a previous CT infection. For the polymorphism analysis, buccal cells were collected and the extracted DNA was analyzed by polymerase chain reaction (PCR), endonuclease digestion and gel electrophoresis using primer pairs specific for the polymorphic region. All material was maintained at -80 ° C and sent on dry ice to the Division of Immunology and Infectious Diseases at Weill Cornell Medicine in New York. Associations between MBL genotypes or alleles and tubal permeability were analyzed by the Pearson chi-square test with or without Yates correction. Results: There was no difference in CT antibody detection between the two groups. Women with obstructed tubes had a higher prevalence of being positive for the heterogenous genotype AB (36%) versus (16%) (p < 0.01). Similarly, the distribution of the normal A allele and variant B allele were also significant different between the two groups (p < 0,01). Conclusion: The findings suggest that while exposure to CT was similar in both groups of women the presence of the variant MBL B allele increases the risk for development of tubal obstruction, subsequent to a CT or other infection. In the Brazilian women evaluated possession of this genetic polymorphism increased the likelihood that blocked fallopian tubes will be a consequence of a genital tract infection

Cryptococcus sp é um fungo saprófita, cosmopolita, que causa micose sistêmica, geralmente, subaguda ou crônica, conhecida, sobretudo, por sua localização meníngea, após aquisição da infecção por via respiratória Embora seja ubíquo, a criptococose ocorre predominantemente em indivíduos imunodeficientes e podendo ocorrer, também, em indivíduos imunocompetentes. Os estudos experimentais e em humanos avaliando a ativação do sistema complemento e a produção de anticorpos específicos mostram que a resposta inata e de anticorpos são importantes para a delimitação do processo infeccioso por Cryptococcus sp, como também, a administração de anticorpos monoclonais podem induzir uma resposta eficaz na disseminação da doença. O sistema complemento contribui para a defesa do organismo contra o Cryptococcus sp de diferentes maneiras: secretando opsoninas e fatores quimiotáticos e colaborando com a ação dos anticorpos específicos, aumentando a interação entre a imunidade inata e adquirida. Os anticorpos antiglicuroxilomanana (GXM) possuem numerosas atividades biológicas: a) opsonização para fagocitose, b) ativação da via clássica do complemento resultando na deposição precoce de fragmentos de C3 no fungo, c) supressão do excesso de acúmulo de C3 pela via alternativa; d) facilitação do clareamento do GXM do soro in vivo, resultando no maior acúmulo de GXM nos tecidos ricos em células do sistema fagocítico mononuclear; e) proteção em modelos murinos da criptococose e f) facilitação de vários aspectos da imunidade celular ao Cryptococcus sp. O objetivo desse estudo foi avaliar a resposta humoral ao GXM e às proteínas da parede celular (Ag S) avaliando a atividade do sistema complemento como também a produção de anticorpos específicos em amostras séricas de adultos com e sem neurocriptococose. Foram coletadas 106 amostras de soro e divididas em 3 grupos: grupo 1- 21 indivíduos com neurocriptococose e baixa exposição a levedura, grupo 2- foi composto por 23 indivíduos saudáveis com alta exposição ao fungo e HIV negativos, granjeiros da cidade de Jumirim localizada a 164 km de São Paulo, na região de Sorocaba e, o grupo 3- 60 indivíduos saudáveis, HIV negativos e com baixa exposição ao Cryptococcus sp. Dois pacientes foram excluídos do estudo por apresentarem tumores (timona e câncer de pulmão). O sistema complemento foi avaliado por ensaio hemolítico (CH 50 e AP 50) e, a dosagem da proteína ligadora de manose (MBL) foi feita por ELISA. Os valores de CH 50 estiveram dentro da normalidade em 17/21, 13/23, 59/60 indivíduos dos grupos 1, 2 e 3 respectivamente. A média dos valores de CH 50 foi diferente significativamente entre o três grupos (P < 0,0001). O grupo 2 mostrou níveis reduzidos significantes em comparação aos dois outros grupos. Os valores de AP 50 estiveram dentro da normalidade em 11/21; 21/23 e 60/60 indivíduos dos grupos 1, 2 e 3 respectivamente. Houve diferença nos valores de AP 50 (P = 0,0005) e apenas um paciente do grupo 1 apresentou valores indetectáveis desta via. Houve diferença significante na dosagem de MBL entre os três grupos (P = 0,0277). Anticorpos IgG anti-GXM foram quantificados por ELISA e expressos por densidade óptica (DO). IgG anti GXM foi detectado em todos os grupos com diferença significante entre eles (P= 0,0127). As médias de IgG anti- GXM (DO) foram: 1.191 (0,49 a 1.217) no grupo 1, 1.572 (0,815 a 2.479) no grupo 2 e 0,965 (0,321 a 1.295) no grupo 3. Dois indivíduos assintomáticos do grupo 2 tiveram títulos de GXM detectáveis (1/256 e 1/32). Quatro pacientes com neurocriptococose faleceram (19%) e seus resultados mostravam: CH 50 normal, 2/4 tinham valores de AP 50 baixo (12 UI/mL) e indetectável; 3/4 tinham altos níveis de MBL e apenas um tinha baixa DO de IgG anti-GXM. Baseado em nosso estudo, podemos concluir que a resposta humoral (sistema complemento e anticorpos) não é suficiente para explicar a susceptibilidade a neurocriptococose, porém a alta e constante exposição ao Cryptococcus sp pode prevenir o desenvolvimento de doença, ou seja, a constante e intensa exposição ao fungo induz a produção de anticorpos que previnem a doença clínica mas não a infecção. Por outro lado fatores genéticos que determinam as concentrações de MBL podem influenciar na susceptibilidade a neurocriptococose. Os anticorpos contribuem para o clearence de GXM, entretanto as concentrações séricas não se correlacionam com resistência à doença / Cryptococcus sp is a fungal pathogen with a worldwide distribution. Although it is ubiquitous in the environment, cryptococcal disease occurs predominantly in immunocompromised hosts and can also occur in apparently immunocompetent individuals. The innate immunity is of special relevance for the antifungal reaction, as it allows an immediate reaction and recognizes a broad variety of fungal pathogens. The host immune response is a major determinant of the outcome of cryptococcal infection; however, the antibodies response is poorly understood. In addition, most of the studies are experimental and there is restricted knowledge concerning the human immune response. Complement system has soluble factors, restrictive regulator proteins and cellular receptors involved in defense mechanism. Glucuroxylomannan (GXM) monoclonal antibodies (MAbs) have numerous biological activities: a) opsonization for phagocytosis, b) activation of the classical complement pathway leading to early deposition of C3 fragments on the yeast, c) suppression overall accumulation of C3 via the alternative pathway; d) clearance facilitation of GXM from serum in vivo, leading to increased accumulation of GXM in tissues rich in mononuclear phagocyte system; e) protection in murine models of cryptococcosis and f) facilitation of various aspects of cellular immunity to Cryptococcus sp. The goal of our study was to evaluate if the antibody response to GXM and cell wall proteins regarding specific antibodies as well as complement system in sera of immunocompetent adults with and without neurocryptococcosis. The aim of our research was to evaluate classical and alternative complement system pathway, to quantify mannose-binding lectin (MBL) as well antibody response to GXM and cell wall proteins (AgS) regarding specific antibodies in sera of immunocompetent adults with and without neurocryptococcosis. One hundred and six samples were collected and classified in 3 groups: group 1- 21 individuals with neurocryptococcosis and low exposure to the yeast; group 2- was composed by 23 healthy individuals, chicken farmings from Jurumirim, a town 164 km to São Paulo, and with high exposure to Cryptoccocus spp and HIV negative. The third group included 60 healthy HIV negative individuals with presumed low exposure to Cryptococcus. Two patients were excluded by report of previous malignancies (timoma and pulmonary cancer). The complement system was evaluated by hemolytic assay and ELISA to MBL. CH 50 and AP 50 values were within the normal range in 17/21; 13/23; 59/60 patients in groups 1, 2 and 3 respectivelly. Mean CH 50 values were significantly different among the three groups (P < 0,0001). Group 2 showed significantly reduced levels in comparison with groups 1 and 3. AP 50 values were within the normal range in 11/21; 21/23; 60/60 patients in groups 1, 2 and 3 respectivelly. There was difference in the AP 50 values (P=0,0005) and one no activation of this pathway in group 1. There was significant difference in MBL among the groups (P = 0,0277). GXM antibodies IgG was measured by ELISA and expressed as optical density (OD). GXM- IgG was detected in all the groups with significant difference among them (P = 0,0127). The means of IgG anti-GXM (OD) were: 1.191 (range 0,49 to 1.217) in group 1, 1.572 (range 0,815 to 2.479) in group 2 and 0,965 (range 0,321 to 1.295) in the group 3. Two of the group 2 individuals had low GXM titers (1/256 and 1/32) and no symptoms. Four patients (4/21; 19%) with neurocryptococcosis died and the results showed: normal classical pathway activation, 2/4 had low (12 UI/mL) or undetectable alternative pathway values ; 3/4 had high MBL concentrations and only one had low OD for IgG anti-GXM. In conclusion, our results suggest that constant and high exposure to Cryptococcus sp can prevent the development of cryptococcosis, i.e. constant and intensive fungal exposition induces protective antibodies to clinical disease but not to the infection. In the other side, genetic factors which determine MBL concentrations could influence the susceptibility to neurocryptococcosis. The antibodies contribute to GXM clearance, however, the concentrations did not correlate with the resistance to the disease