This dissertation is a compilation of published works and a manuscript that seek to understand the possible role of polyamines in the regulation of capsule in Streptococcus pneumoniae (Spn, pneumococcus). Spn remains a major health risk worldwide while the capsule is widely recognized as the principal virulence factor. Polyamines on the other hand are small hydrocarbon molecules known to regulate a number of cellular processes in bacteria. This work investigates the impact of deletion of polyamine biosynthesis gene, SP_0916 (cadA, lysine decarboxylase at the time of first and second publication), on protein expression and the capsule biosynthesis of virulent pneumococcal serotype 4 (TIGR4). We identify loss of capsular polysaccharide (CPS) in the deletion strain and based on proteomics results, we hypothesized that a shift in metabolism that favors the pentose phosphate pathway (PPP) over glycolytic pathway, that could reduce the availability of precursors for CPS had occurred. Comparison of transcriptomic and untargeted metabolomics profile of ∆SP_0916 with TIGR4 shows impaired glycolysis and Leloir pathways that provide CPS precursors, in the mutant strain. Furthermore, gene expression changes indicate possible reduction of common polyamines (cadaverine, putrescine, spermidine and spermine). Targeted metabolomics analysis confirmed reduced levels of polyamines in SP_0916. However, the result suggests that SP_0916 encodes an arginine decarboxylase, contrary to its existing annotation as a lysine decarboxylase in many bioinformatics databases. Biochemical characterization of the purified protein encoded by SP_0916 confirms that it is indeed catalyzes arginine decarboxylation, and exogenous supplementation of agmatine, the product of the reaction, successfully restores capsule biosynthesis. This study fixes an error in annotation of the TIGR4 genome and further establishes the essentiality of agmatine, a product of arginine decarboxylation as the key polyamine molecule modulating pneumococcal capsule. We later compared the impact of deletion of polyamine synthesis by gene deletion (ΔSP_0916) with chemical inhibition of synthesis using α- difluoromethylornithine (DFMO), in multiple pneumococcal serotypes. Results of this dissertation confirmed that pneumococcal pathways impacted by the disruption of polyamine biosynthesis either by gene deletion or chemical intervention are conserved and could regulate capsule synthesis.

Lower respiratory tract infectious diseases are a world-wide healthcare burden with bacterial pathogens accounting for a large portion of primary and secondary infections. The human respiratory tract is home to hundreds of species of microbes that comprise the human airway microbiome. These commensals play a crucial role in human health in part by providing colonization resistance against pathogens. In a previous study from the Surette lab it was shown that specific bacterial isolates from the respiratory microbiome inhibits the growth of pathogens aerobically. This included an isolate of Staphylococcus aureus which inhibited the growth of Enterococcus faecium. This activity was further characterized in this thesis and the underlying mechanism was explored through comparative genomics. As well, this observation provided proof-of-concept for a large-scale screen for additional isolates which inhibit pathogen growth. I hypothesized that the respiratory tract microbiota included many other bacteria capable of inhibiting the growth of respiratory tract pathogens in both aerobic and anaerobic environments, and that anaerobic conditions will identify new activities not detected aerobically. To examine and identify potential beneficial bacteria, I have screened ~5000 respiratory tract bacteria from the Surette lab’s airway isolate collection against four pathogens: Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. The respiratory tract commensals were pinned onto the pathogen-lawn and their interaction was expressed as zones of clearing or altered growth phenotypes of the pathogen. The results of the screen showed that anti-pathogen activity was a common feature of respiratory tract commensals. In particular, S. pneumoniae was inhibited by taxonomically diverse members of the microbiota representing three phyla (Proteobacteria, Firmicutes and Actinobacteria). Many of the facultative anaerobes that inhibited S. pneumoniae expressed their activity in anerobic conditions. / Thesis / Master of Science (MSc) / The human respiratory tract harbours commensal and pathogenic bacteria, and the latter cause most of the lower respiratory tract infections. The commensal bacteria help to train the immune system and impede the growth of pathogens through colonization resistance. A previous study by the Surette lab identified bacterial isolates from the respiratory tract that inhibit the growth of select pathogens, among them, a particular strain of Staphylococcus aureus. Based on the results of the earlier study, I hypothesized that the respiratory tract bacteria is a good source of commensals that can inhibit the growth of S. aureus and other respiratory pathogens, such as Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae. To find potential therapeutic bacteria, I screened ~5000 respiratory tract isolates from the Surette lab’s strain collection for the ability to impair growth of target pathogens. Additionally, I further characterized the activity of the previously identified S. aureus strain against various Lactobacillalles strains and used comparative genomics to identify potential biosynthetic genes required for biosynthesis of molecules with antibacterial activity within the genome of S. aureus. The research reported in this thesis demonstrates that many commensal bacteria that live within our airways have the ability to inhibit the growth of bacterial pathogens. This work may provide a new source of antibiotics against respiratory infections and new strategies to reduce susceptibility to infections in vulnerable populations.

Streptococcus pneumoniae (pneumococcus), a gram-positive bacterium, is commensal to the human nasopharynx. It is also a common cause of respiratory tract infections and multiple invasive diseases worldwide. Pneumococci attach to the nasopharynx, lung, and vascular endothelial cells, which contributes to colonization as well as to the development of pneumonia, bacteremia, and meningitis. Choline binding proteins (CBPs) are a unique set of cell wall proteins conserved within pneumococci. CBPs bind noncovalently to the phosphocholine of the cell wall through choline binding domains. The choline binding domains of the CBPs are highly conserved; however, they are diverse in their affinities and functions due to differences in their functional domains. Several of the CBPs are predicted to play a role in adherence and colonization, though direct evidence of binding to epithelial receptors is lacking. This project focuses on the ability to express and purify some of the lesser characterized CBPs which are predicted to serve as adhesins and to identify their cognate ligand proteins on host cells with the hope of identifying novel bacterial-host interactions that contribute to colonization.

No description available.

La bactérie Streptococcus pneumoniae peut provoquer de sérieuses pathologies chez l'homme telles que des pneumonies, méningites ou septicémies. L'étude de cette bactérie constitue donc un enjeu de santé publique international. Ces dernières années, il a été mis en évidence que les bactéries exprimaient des Sérine/Thréonine Protéine‐Kinases de type eucaryote (STPKs) et que ces dernières intervenaient dans la régulation de nombreux processus cellulaires. Une approche prometteuse serait donc de cibler les mécanismes de régulation contrôlés par les STPKs pour lutter contre les infections à pneumocoque. L'analyse du génome de S. pneumoniae a montré que cette bactérie possède un seul gène codant pour une STPK, la protéine StkP. Mes travaux de thèse ont montré que StkP est un acteur majeur de la division cellulaire et de la morphogenèse du pneumocoque. J'ai montré que son activité kinase est dépendante de la protéine GpsB et qu'elle phosphoryle spécifiquement plusieurs protéines dont la protéine de division DivIVA. L'ensemble de mes travaux permet de proposer un modèle dans lequel la triade StkP/GpsB/DivIVA régulerait finement la division et l'élongation cellulaire du pneumocoque. À plus long terme, ces travaux pourront servir de base à des études plus structurales pour développer des molécules bloquant les processus dépendants de la phosphorylation assurée par StkP, et générer ainsi de nouvelles molécules affectant le pouvoir pathogène du pneumocoque / The bacterium Streptococcus pneumoniae is the causative agent of several diseases such as pneumonia, meningitis or septicemia. The study of this bacterium represents thus an international health challenge. Over the last decade, bacteria have been shown to produce eukaryotic‐like Serine/Threonine Protein‐Kinases (STPKs) that are involved in the regulation of several cellular processes. A promising approach would be to target the regulatory mechanisms controlled by STPKs to combat pneumococcal infections. The pneumococcus possesses a single gene encoding for a STPK, the protein StkP. The aim of my work was to characterize the biological function of StkP. My work shows that StkP plays crucial roles in the cell division and morphogenesis of S. pneumoniae. I show that the cell division protein GpsB is required for the kinase activity of StkP that, in turn, specifically phosphorylates the cell division protein DivIVA. Altogether, I propose a model in which the StkP/GpsB/DivIVA triad finely tunes S. pneumonia cell division and elongation. These data could provide the basis for future structural studies to develop specific inhibitors of StkP‐mediated phosphorylation and affecting pneumococcal virulence

Bakterielle Pneumonien sind weltweit eine der häufigsten Todesursachen und S. pneumoniae ist das häufigste klinische Isolat. Neutrophile Granulozyten gehören zur Klasse der myeloiden Zellen und sind eine wichtige Komponente der angeborenen Immunität gegen bakterielle Infektionen. Krüppel-like factor 4 (KLF4) spielt dabei nicht nur eine Rolle in der Differenzierung der Zellen des Immunsystems, sondern auch während der Infektion bei der Vermittlung inflammatorischer Signale in unterschiedlichen Zelltypen. Diese Studie zeigt zum ersten Mal in vivo, dass myeloides KLF4 Einfluss auf den Krankheitsverlauf hat und die mit einer bakteriellen Pneumonie einhergehende Entzündungsreaktion reguliert. Die hier aufgeführten Ergebnisse demonstrieren, dass der Transkriptionsfaktor KLF4 während einer Pneumokokken Pneumonie in humanen und murinen neutrophilen Granulozyten induziert wird. Diese Induktion ist Zeit- und Dosisabhängig. Außerdem wird die Expression von myeloidem KLF4 durch die Autolyse von S. pneumoniae reguliert, aber nicht über Toll-like Rezeptor 2 (TLR2), TLR4 oder TLR9 vermittelt. Studien in einem Maus-Pneumonie Modell zeigen, dass myeloides KLF4 einen proinflammatorischen Phänotyp bewirkt. Mäuse mit einem KLF4 knockout (KLF4-/-) in myeloiden Zellen haben im Vergleich zu Wildtyp (KLF4+/+) Mäusen eine höhere Bakterienlast in Lunge, Blut und Milz. Obwohl die Produktion proinflammatorischer Zytokine (wie TNF-α, IL-1β und KC) in BALF und Plasma von KLF4-/- Mäusen geringer war, gab es keine Unterschiede bei der Zellrekrutierung in der BALF von KLF4-/- und KLF4+/+ Mäusen. Allerdings war die Zellrekrutierung im Blut der KLF4-/- Mäuse geringer als bei den KLF4+/+ Mäusen. Außerdem wurde eine erhöhte vaskuläre Permeabilität verbunden mit perivaskulären Ödemen und Pleuritis bei KLF4-/- Mäusen während der S. pneumoniae-induzierten Infektion beobachtet. Diese Mäuse erreichten auch eher die humanen Endpunkte als die vergleichbaren KLF4+/+ Mäuse. / Bacterial pneumonia is one of the leading causes of death worldwide. Streptococcus pneumoniae is the most frequently isolated pathogen from clinical pneumonia samples. Neutrophils belong to the class of myeloid cells and forms an important component of this innate immune system against bacterial infections. Krüppel-like factor 4 (KLF4) has been reported to not only play a role in differentiation of cells of the immune system but also in mediating inflammatory signals in different kinds of host cells during infection. This study shows myeloid KLF4 has an impact on pneumococcal pneumonia outcome and regulates the inflammation associated with bacterial pneumonia in vivo in mice. The results presented in the work show that the transcription factor KLF4 is induced in human and mice neutrophils during pneumococcal pneumonia. The induction of KLF4 is time and dose dependent. Additionally, the expression of myeloid KLF4 is regulated by the autolysis of S. pneumoniae but is not mediated via Toll-like receptor (TLR) 2, TLR4 or TLR9. Studies using a mouse pneumonia model showed that myeloid KLF4 exhibits a pro-inflammatory phenotype. Mice with KLF4 knockout (KO) or KLF4-/- in myeloid cells had higher bacterial load in their lungs, blood and spleen in comparison to wildtype (WT) or KLF4+/+ mice. Although there was less pro-inflammatory cytokine (such as TNF-α, IL-1β and KC) production in the broncho-alveolar lavage fluid (BALF) and plasma of KLF4-/- mice yet there no differences in cell recruitment in the BALF of the KLF4-/- and KLF4+/+ mice. There was however less cell recruitment in the blood of KLF4-/- mice in comparison to KLF4+/+ mice. Additionally, an increased vascular permeability associated with perivascular edema and pleuritis was seen during Streptococcus pneumoniae-induced infection in KLF4-/- mice, which also reached earlier the human endpoints than the KLF4+/+ mice.

Introdução: Os receptores Fc são proteínas de importância crucial no processo saúde-doença. São responsáveis pela ativação de mecanismos efetores e modulam a resposta imune e inflamatória. Têm papel central na patogênese de doenças autoimunes, sepse e doenças neoplásicas. O lúpus, protótipo das doenças autoimunes e a sepse, infecção grave que causa disfunção orgânica, são doenças inflamatórias nas quais o papel dos receptores Fc vêm sendo desvendados. Essas patologias têm alta morbidade e mortalidade, impondo enormes custos para sociedade. A descoberta que a E. coli se liga ao receptor CD16 (FcGamaRIII) para evadir-se do sistema imune, através da ligação com a proteína wzxe presente em sua membrana, torna esse receptor um alvo terapêutico interessante. O CD16 é um FcGamaR com ITAM que classicamente tem função ativadora e gera respostas inflamatórias ao se ligar a imunocomplexos. Porém, na sepse, a ligação direta com a E. coli induz uma ativação ITAMi, que bloqueia a produção de ROS e inibe a fagocitose e a morte desta bactéria. A manipulação dessa ativação inibitória (ITAMi), aparentemente anti-inflamatória, pode ser uma estratégia efetiva para o tratamento de doenças inflamatórias como a sepse e o lúpus. Inicialmente visamos avaliar a importância fisiológica e o papel terapêutico do peptídeo ligante do CD16, em modelo de sepse e em modelo de lúpus induzido por pristane, respectivamente. De maneira similar ao CD16, o CD89 (FcAlfaRI) é capaz de mediar uma sinalização dual, ativatória ou inibitória, que depende da forma como se dá sua ligação às imunoglobulinas e imunocomplexos. Por isso, num segundo momento, averiguamos se o FcAlfaRI (CD89) poderia se ligar à bactérias de maneira direta, na ausência de ligantes cognatos e mediar respostas pro ou anti-inflamatórias, protegendo ou não o hospedeiro. Metodologia: Camundongos C57Bl/6, WT e CD16KO com lúpus induzido por pristane, foram tratados com o peptídeo CYWGGTEGAC(IRG Bioscience,USA). A expressão gênica e protéica de diversas citocinas, assim como genes associados a assinaturas de interferon foram avaliados nos pulmões desses animais. Utilizamos também um modelo de sepse através da injeção intra-peritoneal de E. coli WT e E. coli mutante wzxe -/-, no qual avaliamos mortalidade e produção de citocinas. Realizamos experimentos in vitro com BMM e BMDC murinos, fagócitos humanos e bactérias. A expressão de CD89 e de receptores cognatos foi avaliada através de citometria de fluxo. Empregamos a citometria de fluxo com imagem para análise da fagocitose. Foram realizados também, experimentos in vivo com camundongos WT e transgênicos: CD89tg, CD89R209Ltg CD89tgCD16KO, CD16KO e PCRKO. Comparamos mortalidade, produção de citocinas, quantidade de bactérias e lesão tecidual em modelos de CLP e de pneumonia por administração nasal de S. pneumoniae. Produção de ROS pelos BMM foi avaliada por microscopia confocal e, nos PMN, por quimioluminescência. Imunoprecipitação e immunoblotting foram utilizados para avaliar recrutamento de syk e SHP-1. Utilizamos ELISA para ensaios de ligação de bactérias com CD89 e para quantificar TNF-Alfa, IL-1 e IL-6. Resultados: Camundongos injetados com a bactéria mutante wzxe-/- sobreviveram mais e produziram quantidade menor de citocinas reforçando o papel chave da proteína wzxe, no mecanismo de evasão imune da E. coli. Camundongos WT e CD16KO com lupus induzido por pristane, tratados ou não com peptídeo CYWGGTEGAC, não apresentaram diferenças na expressão gênica nem protéica de citocinas nem em genes associados a assinaturas de interferon em seus pulmões. O CD89 interage diretamente com bactérias gram-positivas e gram-negativas. A interação bactéria-CD89 em macrófagos murinos induz ativação celular, fagocitose e morte bacteriana, que são dependentes da cadeia FcRGama. Essa mesma interação protege contra a mortalidade em dois modelos de sepse (CLP e pneumonia) e é dependente da cadeia FcRGama e indepedente de PCR e IgA anti-bactéria. Conclusões: O CD16 e o CD89 são FcRs com ITAM que apresentam uma dualidade na forma de ativação através do ITAM, que em algumas situações pode ser inibitória (ITAMi). Nessa tese reforçamos o papel chave da proteína wzxe, ligante do CD16, como responsável pela evasão bacteriana da E. coli através de sinalização ITAMi. Por outro lado, falhamos em demostrar diferenças após o tratamento de camundongos com lúpus induzido por pristane usando o peptídeo CYWGGTEGAC. Acreditamos que isso tenha ocorrido devido a dose inadequada do peptídeo ou proteólise por enzimas endógenas do camundongo, assim que o peptídeo é injetado. Novas doses ou a manipulação da estrutura do peptídeo são perspectivas futuras para este projeto. Quanto ao CD89, provamos seu papel extremamente importante na imunidade inata. Esse receptor, à semelhança do CD16, foi capaz de ligar-se diretamente a bactérias, na ausência de opsoninas e ligantes cognatos. O CD89 foi protetor tanto para infecção por gram-positivo quanto por gram-negativo enquanto o CD16 foi protetor apenas em modelo de pneumonia por gram-positivo / Introduction: Fc receptors are proteins of crucial importance in the health-disease process. They are responsible for the activation of effector mechanisms and modulate the immune and inflammatory responses. They play a central role in the pathogenesis of autoimmune diseases, sepsis and neoplastic diseases. Lupus, a prototype of autoimmune diseases and sepsis, a serious infection that causes organ dysfunction, are inflammatory diseases in which the role of Fc receptors has been unraveled. These pathologies have high morbidity and mortality, imposing enormous costs for society. E. coli has been found to bind directly to the FcGammaRIII(CD16) receptor to evade the immune system. This is due to the binding to the wzxe protein present in its membrane, making this receptor an interesting therapeutic target. CD16 is an FcGammaR with ITAM that classically has an activating function and generates inflammatory responses when binding to immunocomplexes. However, in sepsis, direct binding with E. coli induces an ITAMi activation, which blocks ROS production and inhibits phagocytosis and death of this bacterium. The manipulation of this apparently anti-inflammatory inhibitory signaling (ITAMi) may be an effective strategy for the treatment of inflammatory diseases such as sepsis and lupus. Initially we aimed to evaluate the physiological importance and therapeutic role of the CD16 binding peptide, in a sepsis model and pristane-induced lupus model, respectively. Similarly to CD16, CD89 (FcAlphaRI) is capable of mediating dual, activating or inhibitory signaling, which depends on how it binds to immunoglobulins and immunocomplexes. Therefore, we assessed whether FcAlphaRI (CD89) could bind to bacteria directly, in the absence of cognate ligands and mediate pro or anti-inflammatory responses, protecting or not the host. Methods: C57Bl/6 mice, WT and CD16KO with pristane-induced lupus were treated with the peptide CYWGGTEGAC (IRG Bioscience, USA). Gene and protein expression of cytokines, as well as genes associated to interferon signatures were evaluated in the lungs of these animals. We also used a sepsis model through the intra-peritoneal injection of E. coli WT and E. coli mutant wzxe-/-, in which we evaluated mortality and production of cytokines. We performed in vitro experiments with murine BMM and BMDC, human phagocytes and bacteria. Expression of CD89 and cognate receptors was assessed by flow cytometry. Flow cytometry with imaging was employed for phagocytosis analysis. In vivo experiments were also performed on WT and transgenic mice: CD89tg, CD89R209Ltg CD89tgCD16KO, CD16KO and CRPKO. We compared the mortality, cytokine production, amount of bacteria and tissue injury in CLP and pneumonia by nasal administration of S. pneumoniae. ROS production by BMM was evaluated with confocal microscopy and, in PMN, by chemiluminescence. Immunoprecipitation and immunoblotting were used to evaluate recruitment of syk and SHP-1. We used ELISA for binding assays with CD89 and bacteria and quantification of TNF-Aphla, IL-1 and IL-6. Results: Mice injected with wzxe-/- mutant E. coli survived more and produced smaller amounts of cytokines, reinforcing the key role of the wzxe protein in the mechanism of immune evasion of E. coli. WT and CD16KO pristane induced lupus mice, treated or not with the peptide CYWGGTEGAC didn\'t show differences in gene or protein expression of cytokines nor in interferon signature genes in their lungs. The bacterial-CD89 interaction in murine macrophages induces cellular activation, phagocytosis and bacterial death, which are dependent on the FcRGamma chain. This same interaction protects against mortality in two models of sepsis (CLP and pneumonia) and is dependent on the FcRGamma chain and independent of PCR and IgA anti-bacterium. Conclusions: CD16 and CD89 are ITAM-bearing FcRs that present a duality in the form of activation through ITAM, which in some situations may be inhibitory (ITAMi). In this thesis we reinforce the key role of wzxe protein, a CD16 ligand, as responsible for the bacterial evasion of E. coli through ITAMi signaling. On the other hand, we failed to demonstrate differences after treatment of pristane-induced lupus mice using the CYWGGTEGAC peptide. We believe that this was due to inadequate dose of the peptide or proteolysis by endogenous mouse enzymes, so the peptide is injected. New doses or manipulation of the peptide structure are future prospects for this project. As to CD89, we proved the extremely important role of CD89 in innate immunity. That receptor, similarly to CD16, was able to bind directly to bacteria, in the absence of opsonins and cognate ligands. CD89 was protective for both gram-positive and gram-negative infection while the CD16 was protective only in a model of gram-positive pneumonia

Introdução: A vacina pneumocócica é recomendada para crianças com IRC, pelo maior risco de adquirir a forma invasiva da doença. O Streptococcus pneumoniae é o principal agente etiológico causador de pneumonia e otite média aguda, e a segunda causa de meningite na criança. A vacina conjugada 7 - valente tem se mostrado imunogênica e com efeito duradouro em crianças normais, porém em pacientes com IRC, devido às alterações imunológicas associadas à uremia, pode resultar uma resposta sub - ótima e de vida curta. Objetivo: Determinar as concentrações de anticorpos da classe IgG contra os sorotipos 4,6B, 9V, 14, 18C, 19F e 23 F do Streptococcus pneumoniae, antes e depois da administração da vacina antipneumocócica conjugada 7 - valente, em crianças portadoras de insuficiência renal crônica em tratamento conservador e diálise, e comparar a resposta entre eles. Métodos: Selecionamos 48 crianças, com idade de 1 ano a 9 anos, portadoras de IRC, com depuração de creatinina menor ou igual a 70 ml/min/1,73m 2 , calculada pela fórmula de Schwartz, divididos em: grupo 1 (G1, tratamento conservador), e grupo 2 (G2,tratamento dialítico). Os pacientes receberam duas doses da vacina conjugada 7 - valente, com intervalo de 60 dias. A sorologia foi colhida antes da primeira dose, e 30 a 60 dias após o reforço. Os títulos de anticorpos para os sorotipos presentes na vacina (4, 6B, 9V, 14, 18C, 19F, 23F) foram determinados pelo método imunoenzimático (ELISA). Resultados: A análise da concentração pré - vacinal de IgG mostrou maior percentual dos pacientes do grupo 1 e 2 com IgG <0,6 para os sorotipos 4, 9 e 18, e maior percentual com IgG maior ou igual a 1,3 ug/ml para os sorotipos 14 e 19. Para avaliar a concentração de IgG pós - vacinal foi utilizado o cálculo de freqüências com intervalo de confiança 95%(IC95), para os 7 sorotipos nos 2 grupos, utilizando-se 3 critérios : Critério A: IgG pós vacinal maior ou igual a 1,3 ug/ml, por este critério verificou-se freqüência de resposta do G1 de 0,650 (IC95: 0,407-0,864) a 1,0 (IC95: 0,663 - 1,0) e do G2 de 0,777 (IC95: 0,399 -0,971) a 1,0 (IC95: 0,663 - 1,0), critério B: ou seja, delta (pré/pós) maior ou igual a 4X, freqüência de resposta G1 de 0,458 (IC95: 0,255 - 0,671) a 0,708 (IC95%: 0,488 - 0,873) e do G2 de 0,458 (IC95%: 0,255 - 0,671) a 0,708 (IC95%: 0,488 - 0,973), e critério C:isto é, delta (pré/pós) maior ou igual a 4X e IgG maior ou igual a 1,3 ug/ml, freqüência de resposta do G1 de 0,416 (IC95%: 0,221 - 0,633) a 0,625 (IC95%: 0,406 - 0,811) e do G2 de 0,416 (IC95%: 0,221 - 0,633) a 0,666 (IC95%: 0,447 - 0,843). G1 e G2 mostraram comportamento semelhante na análise das freqüências de resposta, segundo os 3 critérios. Conclusão: Este estudo é pioneiro na avaliação da vacina antipneumocócica conjugada em crianças com IRC, e demonstrou uma boa resposta para cada critério analisado separadamente, com comportamento semelhante nos 2 grupos. A dificuldade inerente à definição de critérios de soroconversão, demonstra a necessidade de estudos multicêntricos com acompanhamento clínico e laboratorial em longo prazo para avaliar a sustentabilidade da soroconversão e a imunogenicidade da vacina. / Introduction: The pneumococcal vaccine is recommended for children with CRF due to their increased risk in acquiring the invasive form of the disease. Streptococcus pneumoniae is the main etiological agent that causes pneumonia and acute middle otitis and the second cause of meningitis in children. The 7-valent conjugated pneumococcal vaccine has been shown to be immunogenic and to have a lasting effect in normal children; however, in children with CRF, the vaccine can produce a suboptimal and short-lasting response due to the immunological alterations associated to uremia. Objective: to determine pneumococcal IgG antibodies to serotypes 4, 6B, 9V, 14, 18C, 19F e 23 F before and after 7 - valent conjugated pneumococcal vaccine, in children with chronic renal failure in conservative treatment and dialysis. Methods: 48 children with CRF, aged 1 to 9 years, with a creatinine clearance of menor ou igual a 70 ml/min/1.73 calculated by the Schwartz formula, were selected for the present study and divided in two groups: Group 1: conservative treatment and Group 2: dialytic treatment. The patients received two doses of the 7- valent conjugated vaccine, with a 60-day interval between them. Serological samples were collected before the first dose and 30 to 60 days after the second one. Antibody titers for the serotypes present in the vaccine (4, 6B, 9V, 14, 18C, 19F, 23F) were determined by the immunoenzymatic method (ELISA). Results: The analysis of the pre-vaccinal IgG concentration showed a higher percentage of patients from Groups 1 and 2 with IgG < 0.6 for the serotypes 4, 9 and 18, and a higher percentage with pre-vaccinal IgG maior ou igual a 1.3 ug/ml for the serotypes 14 and 19. To assess the post-vaccinal IgG concentration, the calculation of frequencies with 95% confidence interval (95CI) was employed for the 7 serotypes in both groups, using three criteria: Criterion A: post-vaccinal IgG maior ou igual a 1.3 ug/ml, this criterion verified the frequency of response from G1 0,650 (IC95: 0,407-0,864) to 1,0 (IC95: 0,663 - 1,0) and G2 0,777 (IC95: 0,399 - 0,971) to 1,0 (IC95: 0,663 - 1,0), criterion B: i.e., delta (pre/post) maior ou igual a 4X, frequency of response from G1 0,458 (95CI: 0.255 ? 0.671) to 0.708 (95CI: 0.488 - 0.873) and G2 0,458 (IC95: 0,255 - 0,671) to 0,708 (IC95: 0,488 - 0,873); criterion C: i.e., delta (pre/post) ?4X and IgG maior ou igual a 1.3 ug/ml, frequency of response from G1 0.416 (95CI: 0.221 - 0.633) to 0,625(95CI: 0,406 - 0,811) and G2 0,416 (9CI: 0,221 - 0,633) to 0.666 (95CI: 0.447 - 0.843). Groups 1 and 2 showed similar behavior at the analysis of response frequencies according to the three criteria. Conclusion: this is the first study to assess the conjugated pneumococcal vaccine in children with CRF, which showed good response, with a similar behavior in both groups. The inherent difficulty in defining criteria for seroconversion demonstrates the need for multicentric studies with long-term clinical and laboratory follow up, in order to assess the seroconversion duration and the vaccine immunogenicity.

Introdução: A Imunodeficiência Comum Variável (ICV) faz parte de um grupo de imunodeficiências primárias na qual os pacientes apresentam defeitos na maturação e diferenciação dos linfócitos B (LB), resultando em distúrbios funcionais além de alterações na distribuição de seus subtipos. Consequentemente, estes pacientes apresentam hipogamaglobulinemia, susceptibilidade a infecções e ausência de produção de anticorpos a antígenos específicos. Na tentativa de reduzir os episódios de infecções recorrentes, alguns trabalhos têm recomendado a vacinação com patógenos mortos ou subunidades e em trabalho anterior demonstramos a eficácia clínica da vacinação de pacientes com ICV, porém, a experiência com a administração de vacinas em imunocomprometidos é limitada. Objetivos: Avaliar a cinética da distribuição das subpopulações de linfócitos B antes e após a vacinação com antígenos proteicos e polissacarídicos em pacientes com ICV acompanhados no Ambulatório de Imunodeficiências Primárias do Hospital das Clínicas, FMUSP, além da produção de anticorpos específicos aos antígenos vacinais. Pacientes e Métodos: Um grupo de 35 pacientes com ICV e 16 controles foram vacinados contra Influenza, H1N1 e S. pneumoniae. Após as coletas nos tempos pré e pós 1, 3 e 6 meses foram realizados a separação de PBMC e cultura de linfócitos com lisado viral e hemaglutinina de Influenza, além da citometria de fluxo para identificação das subpopulações de LB naive, zona marginal (MZB), memória com troca de isotipo (SMB) e plasmoblastos (PBL). Foram dosados os anticorpos específicos e no grupo dos pacientes foi aplicado um score de sintomas antes e após a imunização. Resultados: Apesar da redução significativa na pontuação do score de sintomas, a maioria dos pacientes não produziu anticorpos específicos para Influenza, H1N1 e S. pneumoniae. A análise da cinética das subpopulações de LB revelou que em indivíduos saudáveis, a resposta contra Influenza apresentou duração de 6 meses, observada por meio da redução da subpopulação naive e aumento gradual da frequência de SMB a partir do primeiro mês. Observamos também redução da população de memória por volta do 3º mês, com aumento da população de PBL que permaneceu elevada até o 6º mês. Por outro lado, a despeito de os pacientes apresentarem aumento de SMB no primeiro mês após a vacinação, sua frequência foi inferior ao observado nos controles, decaindo ao terceiro mês. A população de PBL apresentou aumento precoce no primeiro mês após a vacinação, também muito menor do que observado nos controles, não sendo mantido no terceiro mês. Ainda, observamos uma correlação entre o aumento da expressão destas duas subpopulações no primeiro mês. Apenas a população de MZB apresentou aumento significativo no terceiro mês nos pacientes quando comparados aos controles. Ao dividirmos os pacientes de acordo com a expressão de SMB e PBL após 1 mês da administração das vacinas, observamos que os pacientes que apresentaram aumento na expressão de células B de memória foram os que exibiram uma melhora clínica mais expressiva, soroconverteram e desenvolveram soroproteção para H1N1.Conclusões: Apesar de não apresentarem eficaz diferenciação em células de memória e efetoras, resultando na resposta precoce e de curta duração, observamos que os pacientes foram capazes de reconhecer e responder às vacinas. Além disso, a elevada expressão de MZB no terceiro mês após a vacinação pode sugerir a atuação desta subpopulação na apresentação para os LT. Estes achados reforçam a necessidade de uma melhor compreensão da ativação do sistema imune em pacientes com ICV, para uma adequada subdivisão de acordo com o perfil de resposta após a vacinação / Introduction: Common Variable Immunodeficiency (CVID) is a primary antibody deficiency characterized by defects in B lymphocyte maturation, resulting in disturbed differentiation, distribution and functional variations on its subtypes. As a result , CVID patients have hypogammaglobulinemia and poor antibody response to specific antigens with increased susceptibility to infections. In an effort to minimize the recurrent episodes of infections, some studies have recommended immunization with inactivated pathogens or subunits and in a former study we have shown the clinical improvement determined by immunization in CVID patients, but the experience with vaccines\' administration to immunodeficient patients is limited. Objectives: To evaluate the changes in distribution of B cell subtypes before and after vaccination of CVID patients followed at the Division of Clinical Immunology and Allergy of University of São Paulo Medical School with protein and polysaccharide antigens, as well as specific antibody production . Methods: A group of 35 CVID patients and 16 controls were vaccinated against Influenza, H1N1 and S. pneumoniae vaccines. Blood samples were collected before and 1, 3 and 6 months post vaccination. PBMCs were stimulated with Influenza viral lysate and hemagglutinin peptide. Flow cytometry was performed to identify naïve B cells, marginal zone (MZB), switched memory B cells (SMB) and plasmablasts (PBL). Specific antibody production was measured and a symptoms score was applied for clinical evaluation before and after immunization. Results: In spite of the significant reduction in symptoms score after vaccination, most patients didn\'t produce specific antibodies to Influenza, H1N1 and S. pneumoniae. The analyzes of B cell subtypes changes in healthy individuals upon in vitro Influenza stimulation showed that the response endured up to 6 months post immunization. We observed a reduction in naïve B cell frequency while gradual increase in SMB frequency occurred already at 1 month after vaccination. Moreover, as the memory cell population declined, PBL population increased at the third month post vaccination until the sixth month. Although patients had an increase of SMB on the first month after vaccination, it was lower than that observed in controls, decreasing by the third month post vaccination. Plasmablast frequency had an early increase on the first month, also much lower than the observed in controls decreasing by the third month. In addition, we observed a correlation between the increased expression of SMB and PBL on the first month post vaccination. In patients, only MZB subtype presented a significant increase on the third month when compared to controls. We divided the patients according SMB and PBL expression after 1 month post vaccination and we observed that patients who were able to produce memory B cells showed a better clinical improvement, developed H1N1 seroconversion and seroprotection. Conclusion: Despite the defect on differentiation into memory and effector B cells resulting in early response with lowduration, we observed that patients were able to recognize and respond to vaccines. In addition, the over expression of MZB on the third month after vaccination may suggest the role of this subpopulation as an antigen presenting cell for T cells. These findings reinforce the need of a better understanding of immune system activation and response in CVID patients to propose a division according to vaccine (antigen) responders and non responders

Die bakterielle Meningitis ist trotz der Anwendung modernster Antibiotika mit einer hohen Letalität und neurologischen Spätkomplikationen verbunden. Ein entscheidendes Ereignis ist dabei der Zusammenbruch der Blut-Hirn-Schranke (BHS). Die genauen Mechanismen, die zu ihrer Schädigung führen, sind bis heute unklar. In dieser Arbeit wurde untersucht, ob lebende Pneumokokken in einem Zellkulturmodell der BHS zu einer apoptotischen Zellschädigung von zerebralen Endothelzellen, als wichtigstem zellulären Bestandteil der BHS, führen und damit zu ihrer strukturellen Schädigung beitragen. Mittels verschiedener Detektionsmethoden (TUNEL, Fluoreszenzmikroskopie, Elektronenmikroskopie) konnte nachgewiesen werden, daß Streptococcus pneumoniae zu einem apoptotischen endothelialen Zelltod führt. Eine Beteiligung von Caspasen konnte weder mit direkter Aktivitätsmessung noch mittels Inhibitionsexperimenten oder dem Nachweis von Caspase-spezifischen Substraten gezeigt werden. Insgesamt sind die Morphologie der Zellkerne und die spezifische Degradation der endothelialen DNS hinweisend für einen Apoptosis-Inducing-Factor-vermittelten Zelltod ohne Caspasenbeteiligung. Diese Form des Zelltodes ist bereits in anderen Zellmodellen, bisher jedoch nicht bei zerebralen Endothelzellen beschrieben worden. Auf Seiten des Bakteriums konnten Wasserstoffperoxid und Pneumolysin als Auslöser der Apoptose identifiziert werden. Die zytotoxische Potenz des Pneumolysins ist dabei an dessen Poren-formende Aktivität gebunden. Die Ergebnisse sind von potentieller klinischer Relevanz, da es bei einer Bakteriämie und während der Invasion der Pneumokokken in das ZNS zu einem direkten Kontakt zwischen Bakterien und zerebralen Endothelzellen kommt und sich daraus eine Möglichkeit zur Entwicklung adjuvanter Therapien ergeben könnte. / Despite sufficient antibiotic treatment, pneumococcal meningitis has remained a disease associated with high mortality and neurological sequelae. The disruption of the blood brain barrier (BBB) is regarded a key event in the initial phase of pneumococcal meningitis. However, the exact molecular mechanisms involved in this process are still unknown. The aim of this study was to determine if living pneumococci are able to induce apoptosis in cerebral endothelial cells - the main cellular component of BBB - and therefore might contribute to its damage. Using several different detection methods (TUNEL, fluorescence and electron microscopy), induction of apoptotic cell death of endothelial cells by pneumococci could be verified. An accompanying activation of caspases was not detectable, despite the use of specific detection techniques such as inhibition experiments, direct enzyme measurements and detection of caspase-specific protein cleavage. These results as well as the specific nuclear morphology and degradation of endothelial DNA suggest an involvement of the mitochondrial protein Apoptosis inducing factor (AIF). This is the first time this specific form of apoptotic, AIF-driven cell death has been described to be engaged in endothelial cells. On the part of the bacterium, pneumolysin and hydrogen peroxide were identified as the two main inducers of apoptosis. The cytotoxic potency of pneumolysin is related to its pore-forming activity. These results are of clinical relevance since pneumococci are known to reside in close proximity to cerebral endothelial cells during bacteriemia and their entry into the CNS. These findings could contribute to the development of adjuvant treatment of bacterial meningitis.