Numerous studies have focused on identifying novel probiotic-based treatment options for immune homeostasis maintenance and favorable modulation of the gut microbiota which acts as a key regulator of the gut-brain axis. Recently, probiotics interventions are gaining interest as effective approaches to treat neuropsychiatric disorders through the gut-brain axis. However, there is limiting knowledge about probiotics' effects during puberty on the developing brain and immune responses. Probiotic intake could offer a strategy to counteract the immune, microbial and behavioral disturbances induced by inflammatory LPS. Thus, we hypothesized that the intake of a novel probiotic bacterium Rouxiella badensis subsp. acadiensis would modulate the immune response and that pubertal administration could mitigate LPS- induced inflammation and prevent enduring behavioral changes later in life. We investigated the interaction of the probiotic with the intestinal mucosa and its ability of modulating the gut mucosal immunity (Article 1). Next, we examined the ability of pubertal treatment with R. badensis subsp. acadiensis to alleviate LPS-induced anxiety-like and depression-like behaviors in adult male and female mice and to affect the expression of 5HT1A receptors in specific brain areas of adult mice (Article 2). We finally studied the ability of R. badensis subsp. acadiensis treatment during puberty to mitigate the effects of LPS on the immune system and on the gut microbiome composition (Article 3). These studies have demonstrated the ability of R. badensis subsp. acadiensis to survive the gastrointestinal conditions, interact with the gut epithelium and modulate the intestinal homeostasis. Pubertal use of the bacterium was associated with sex-specific effects on the acute immune response, microbiome structure, enduring neurobehavioral outcomes and the expression of 5HT1A receptors in specific brain areas, later in life. This dissertation emphasizes on the importance of puberty as a window of opportunities during which probiotic use can alleviate the long-term neural, behavioral, immunological and microbiome alterations induced by stress.

While adenovirus (Ad) vectors have been shown to elicit potent antigen-specific T cell responses, the kinetics and nature of antigen-specific mucosa! and systemic T-cell responses has rarely been examined, especially following mucosal administration of Ad-based vectors. In the present studies, the phenotypic and functional characterization of antigen-specific CD8+ T cell responses following intrarectal (i.r.) vaccination with an Ad vector expressing Gallus gallus ovalbumin (OVA) was conducted. The frequencies of OVA-specific CD8+ T cells was maximal at 2 weeks post-vaccination in all tissues examined and then declined, demonstrating normal expansion and contraction kinetics. CD8+ T cells induced in the course of immunization exhibited phenotypic characteristics of effector memory T cells including up-regulation of the cell surface molecules CD43, CD44 and a low level of expression of CD127 at both local and systemic sites. While the discordance between the number of tetramer-reactive and cytokine-producing OVA-specific CD8+ T cells was observed, CD8+ T cells appeared to be fully functional in vivo. Upon secondary antigen exposure, the CD8+ T cell population expanded dramatically, particularly at the mucosa! surfaces. In addition, the CD8+ T cell response generated in the course of i.r. priming protected mice from intravaginal (i. vag.) vaccinia virus one month after immunization, thus underscoring the importance of inducing a tissue-resident effector memory T cell subset for protection against pathogens at mucosal surfaces. In developing future vaccines for mucosal diseases, the induction of a tissue-resident effector memory T cell subset should be one of the immunization objectives. Lentiviral vectors represent an attractive mode of genetic vaccination. Most commonly used, vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentiviral vectors do not efficiently infect epithelial cells from the apical side, and, therefore, are not suitable as mucosa! vaccines. In the present studies, Ebola Zaïre strain glycoprotein (EboZ)-pseudotyped lentiviral vectors, which have been previously used to deliver transgene to the lung epithelium, were delivered i.r. and evaluated as a mucosal booster vaccine. Rectal delivery of EboZ-pseudotyped lentiviral vectors expressing β-galactosidase (β-gal) had resulted in low, but detectable levels of β-gal expression 2 weeks after administration. When delivered on its own, EboZ-pseudotyped lentivirus did not prime detectable antigen-specific immune response. However, when delivered i.r. 30 days after i.r. Adβ-gal immunization, a significant enlargement (boost) of β-gal-specific CD8+ T cell responses, especially in the colonic lamina propria (LP), was observed as compared to the delivery of EboZ-pseudotyped vector encoding different transgenes or VSVG-pseudotyped lentivirus expressing β-gal. When these animals were i. vag. challenged with vaccinia virus expressing β-gal, a dramatic expansion of β-gal-specific CD8+ T cells, especially in the vaginal tract, was observed. In addition, this prime and boost strategy protected the mice from i. vag. vaccinia virus challenge. Therefore, i.r. Ad-based priming followed by i.r. EboZ-pseudotyped lentiviral boosting was an effective strategy for eliciting protective mucosal CD8+ T cell responses. / Thesis / Doctor of Philosophy (PhD)

Clinical and experimental studies have shown that estradiol (E2) can enhance protection against sexually transmitted infections such as HSV-2 and HIV-1. Antigen presenting cells (APCs) such as Dendritic cells (DCs) are critical for generating immune responses against these infections, and it is unclear whether unique factors present in the genital mucosa can influence immune responses by directly modulating the phenotype and function of local APCs. To address this, I hypothesized that sex hormones, such as E2 and innate factors in the local microenvironment can regulate the phenotype and function of vaginal APCs. The work summarized in this thesis addressed this central hypothesis. In the first section of the thesis, I examined whether vaginal APCs were distinct in their phenotype and function compared to those in other mucosal tissues or spleen. The results show that the vagina was enriched in CD11c+ CD11b+ MHCII− DCs. Functionally, vaginal tissue cells (TC) and CD11c+ DCs were more potent inducers of Th17 responses in co-cultures with CD4+ T cells, compared to lung, small intestine or spleen APCs. E2 was critical for the conditioning of vaginal DCs to prime these Th17 responses through an IL-1-dependent pathway, indicating that sex hormones such as E2 can directly influence the function of vaginal APCs. In the next section, I determined whether other co-factors in the genital microenvironment such as microflora and innate lymphocytes could also influence vaginal APC functions. We found that while microflora was not essential, IL-17 produced by innate lymphocytes was critical for the induction of IL-1 from DCs, and consequently for potentiating Th17 responses. Finally, I attempted to develop an in vivo mouse model where the effect of E2 on vaginal APCs could be examined in the context of genital HSV-2 infection. I tested a 7-day injectable E2 and a 21-day E2 pellet delivery model, and found that both regimes had limitations for examining E2-effects on anti-viral responses. Yet, subsequent to the work done in this thesis, we were able to confirm our observations of E2-conditioned Th17 responses in vivo in an intranasal immunization model utilizing E2 pellet delivery, and thereby addressed the mechanism underlying enhanced anti-viral protection following E2-treatment. In conclusion, this is the first study to show the effect of E2 on genital tract APCs and their ability to prime Th17 responses. It provides future avenues to examine whether modulation of this microenvironment can help optimize vaccine-induced immune responses against STIs. On a more fundamental level, it highlights the need to consider the inherent distinctions in APC populations among different mucosal tissues. / Dissertation / Doctor of Philosophy (PhD)

No description available.

Inflammatory bowel diseases, (IBD), are a group of chronic disorders of the gastro-intestinal tract, and include Crohn’s disease (CD) and Ulcerative Colitis (UC). The pathogenesis is not known, but involves at least in part a loss of tolerance towards the commensal colonic microbiota. In this thesis, we show in animal models of CD and UC that the colonic mucosal blood flow increased compared to healthy animals. This blood flow increase is due to an up regulation of endothelial nitric oxide synthase (NOS). Further, we show in the UC model that the thickness of the firmly adherent colonic mucus layer increased compared to healthy animals. This increase is due to an up regulation of inducible NOS in the epithelium. Both the blood flow and mucus thickness increase appear to be protective mechanisms.  We demonstrate that the firmly adherent colonic mucus layer acts as a partial barrier towards luminal bacteria. In the UC model, this barrier is destroyed, causing increased bacterial translocation. The adhesion molecule P-selectin was up regulated in the UC model, leading to increased interactions between leukocytes and the endothelium, but also increased interactions between platelets and the endothelium. This indicates that not only leukocytes, but also platelets are involved in colonic inflammation. The addition of the probiotic bacterial strain Lactobacillus reuteri prevented disease by normalizing P-selectin levels and endothelial interactions with leukocytes and platelets. Lactobacillus reuteri also decreased bacterial translocation over the epithelium. In summary, this thesis highlights the importance of colonic barrier functions, and investigates the role of the microbiota in experimental IBD.

Dans l'alimentation des pays industrialisés, l'apport en protéines est bien supérieur à l'apport nutritionnel conseillé (ANC). De plus, cet apport peut être encore supérieur lors de la consommation de régimes riches en protéines utilisés à des fins de perte de poids par des personnes obèses ou en surpoids. Cependant, les conséquences des régimes riches en protéines au niveau de l'écosystème du gros intestin sont encore très mal connues. Dans la première partie de cette thèse, nous avons étudié l'impact d'un régime hyperprotéique sur le microbiote, le contenu endoluminal du gros intestin et le métabolisme des colonocytes. Les rats ont consommé pendant 15 jours soit un régime hyperprotéique (53% de protéines) soit un régime normoprotéique (14% de protéines). Nous avons observé que le régime hyperprotéique réduit la quantité des groupes bactériens majeurs comme Clostridium coccoides et Clostridium leptum, ainsi que Faecalibacterium prausnitzii dans le microbiote du gros intestin avec conjointement des modifications sur sa biodiversité. En même temps, les quantités des produits finaux de la fermentation des acides aminés par le microbiote, les acides gras à chaîne courte (AGCC) et les acides gras à chaîne branchée sont fortement augmentées. Cependant, l'expression des transporteurs des acides monocarboxyliques et l'oxydation du butyrate par les colonocytes ne sont pas modifiés en lien avec des modifications mineures des concentrations en AGCC dues à une augmentation des contenus du gros intestin après l'ingestion du régime hyperprotéique. Il en résulte une augmentation de l'excrétion des AGCC dans les fèces. Ces phénomènes permettraient une homéostasie du métabolisme du butyrate dans les colonocytes, en lien avec le rôle crucial de cet AGCC sur l'épithélium du côlon. Dans la deuxième partie de cette thèse, nous avons testé l'effet d'un mélange d'acides aminés (Thr, Met et Glu) sur la cicatrisation de la muqueuse colique après une colite induite par le DSS (dextran sodium sulfate) ; un modèle d'étude des maladies inflammatoires intestinales souvent utilisé. Une optimisation de la cicatrisation de la muqueuse intestinale émerge comme une cible thérapeutique, dans la prise en charge de ces maladies. La colite a été induite chez le rat avec 5% (w/v) de DSS pendant 6 jours, puis, à l'arrêt du traitement DSS, les animaux ont soit reçu le mélange d'acides aminé soit l'Ala comme témoin iso-azoté, pendant 3, 7 et 10 jours. Nous avons observé que 10 jours de complément en mélange d'acides aminés améliorent la cicatrisation post-colite, avec des modifications sur le taux de synthèse protéique dans la muqueuse colique, sans toutefois modifier la résolution de l'inflammation. Nos résultats suggèrent que l'utilisation des mélanges d'acides aminés améliore la cicatrisation de la muqueuse colique après colite chimio-induite. / In industrialized countries, protein intake is largely higher than the recommended dietary allowance (RDA). Furthermore, high protein diets are used for their slimming effect by obese or overweight people. However, little is known regarding to the consequences of a high protein diet on the large intestinal ecosystem. We thus study the influence of a high protein diet on the microbiota, on the endoluminal composition of the large intestine and on the butyrate metabolism by isolated colonocytes. Rats received during 15 days either a high protein diet (53% of proteins) or a normo protein diet (14% of proteins). We observed that the quantity of major bacterial groups Clostridium coccoides and Clostridium leptum, but also Faecalibacterium prausnitzii was reduced in the microbiota of the large intestine together with modifications of its biodiversity. In the same time, the quantities of short-chain fatty acids (SCFA) and branched-chain fatty acids, final products of bacterial fermentation of amino acids, were increased. However, the expression of monocarboxylic acid transporters and butyrate oxidation in colonocytes remained unchanged, in association with minor changes of the SCFA concentrations due to marked increase of the weight of the large intestine content. We then observed an increase in the amount of SCFA in the feces. These phenomena would allow homeostatic metabolism of butyrate in colonocytes, in relationship with its crucial role on the colonic epitheliumIn. In the second part of this thesis, we have tested the effects of a mixture of amino acids (Thr, Met and Glu) on the colonic mucosa healing after colitis induced by DSS (dextran sodium sulphate); a model to study intestinal inflammatory bowel diseases largely used. Optimization of intestinal mucosa healing is more and more considered as a therapeutic goal. Colitis was induced in rats by 5% (w/v) DSS during 6 days, then at the end of the treatment with DSS, animals received either the amino acid mixture or Ala as iso-nitrogenous control, during 3, 7 or 10 days. We observed that 10 days amino acid mixture supplementation was able to improve the colonic mucosal healing, with modification of the protein synthesis rate, without however changes in the resolution of inflammation. Our results suggest that the supplementation with the amino acid mixture improve the mucosal healing after experimental colitis.

Leishmanioses são doenças causadas por protozoários do gênero Leishmania e se apresentam sob forma tegumentar ou visceral. No Brasil, a leishmaniose tegumentar americana (LTA) é causada, na sua maioria, por Leishmania (Viannia) braziliensis e conhecem-se principalmente as formas cutânea (LC), mucosa (LM) e disseminada (LD) da doença. Na LTA as formas clínicas tem sido atribuídas a diferenças na resposta imune do hospedeiro, mas recentemente vinculam-se também à variabilidade intraespecífica da L. (V.) braziliensis. Neste estudo avaliamos se haveria variabilidade biológica nos isolados de L. (V.) braziliensis, provenientes de pacientes com LC, LM e LD, principalmente em resposta a fator de crescimento insulina-símile-I (IGF-I). Os fatores de crescimento do hospedeiro tem sido alvo de estudos no desenvolvimento das leishmanioses, sendo IGF-I um deles. Havíamos demonstrado em estudos anteriores, utilizando Leishmania (Leishmania) amazonensis, que IGF-I induz proliferação, aumentando a atividade da arginase, com geração de poliaminas e diminuindo a síntese de óxido nítrico. No presente estudo analisamos o efeito de IGF-I em L.(V.) braziliensis, espécie prevalente no Brasil. Avaliamos inicialmente as características dos diferentes isolados enquanto promastigota e no prosseguimento enquanto amastigota em células de linhagem monocítica humana THP-1, com e sem estímulo de IGF-I. Nossos dados sugerem que há diferenças na atividade da arginase basal entre os isolados de L. (V.) braziliensis, sendo maior naqueles provenientes de pacientes com LM. IGF-I aumentou a atividade da arginase nos isolados de LC e LD, mas não de LM. Nos isolados em forma amastigota nas células de linhagem monocítica humana THP-1, o efeito de IGF-I foi de aumento do parasitismo nos isolados de LC e LM e de diminuição com os de LD. Nos isolados de LD a atividade da arginase basal foi menor que nos de LC. Por outro lado, a produção de óxido nítrico tendeu a ser maior em isolados de LD quando sob estímulo de IGF-I. Os dados sugerem que diferenças nas características biológicas dos parasitos podem contribuir na apresentação clínica dos casos da LTA. / Leishmaniasis are diseases caused by protozoa of the genus Leishmania that may manifest as cutaneous or visceral disease. In Brazil, American tegumentary leishmaniasis (ATL) is caused mostly by Leishmania (Viannia) braziliensis and cutaneous (CL), mucosal (ML) and disseminated (DL) forms of the disease are known.The diversity of clinical manifestations has been attributed to differences in the host immune response, but recently it has also been related to intraspecific variability of L. (V.) braziliensis. In the present study we evaluated whether there were biological variability in different isolates of L. (V.) braziliensis from patients with CL, ML, and DL, mainly in response to insulin-like growth factor-I (IGF-I). Growth factors of the host have been investigated in the development of leishmaniasis including IGF-I. In previous studies using Leishmania (Leishmania) amazonensis IGF-I was shown to induce proliferation, to increase the activity of arginase, generating polyamines and to decrease the synthesis of nitric oxide. In this study we analyzed the effect of IGF-I in L. (V.) braziliensis, a species prevalent in Brazil. Initially we evaluated the characteristics of individual isolates as promastigote and further as amastigote within human macrophage cell line THP-1 with and without IGF-I stimulation. Our data suggest that there are differences in the basal arginase activity amongst isolates of L. (V.) braziliensis, being higher in those from patients with ML. IGF-I increased the activity of arginase in the isolates of CL and DL, but not of ML. In isolates in the form of amastigotes within THP-1 cells, IGF-I induced the increase of parasitism of isolates from CL and ML, and decrease of those from DL. In isolates of DL the basal arginase activity was lower than in those of CL. Moreover, the production of nitric oxide tended to be higher with isolates of DL upon IGF-I stimulation. The data suggest that differences in the biological characteristics of parasites may contribute to the diversity of clinical presentation of ATL.

Linhagens de Escherichia coli enterotoxigênica (ETEC) são consideradas como importante agente de diarréia, principalmente entre crianças e turistas em países em desenvolvimento. Entre os fatores de virulência expressos por ETEC, as enterotoxinas termo-lábil (LT) e termo-estável (ST) representam os mais relevantes fenótipos. Evidências preliminares sugerem que a severidade da diarréia associada a linhagens de ETEC deve refletir a diversidade natural de linhagens selvagens quanto à produção de enterotoxinas e/ou à ocorrência de variantes naturais com efeitos tóxicos reduzidos. No presente trabalho, investigamos diversidade genética do óperon etx, que codifica para a toxina LT, e da capacidade de produção e secreção de LT por linhagens de ETEC isoladas de humanos ou suínos em diferentes regiões geográficas. Os resultados mostraram considerável variabilidade na produção de LT com valores variando de 2 a 2.525 ng de toxina por mL de cultura. Secreção de LT foi também variável com valores variando de menos que 0,04% a 49,5% do total de LT produzida pelas diferentes linhagens de ETEC. Adicionalmente, experimentos de alça ligada em coelho mostraram uma boa correlação entre a quantidade de LT secretada sob condições in vitro e a capacidade de causar acúmulo de fluidos in vivo. Nós determinamos ainda diversidade de ETEC pela obtenção das seqüências dos óperons etxAB de 50 linhagens (LT+ or LT+/ST+) pertencentes a diferentes sorotipos com ênfase para as linhagens produtoras apenas de LT e isoladas de crianças assintomáticas. As seqüências de nucleotídeo completas dos genes etxAB revelaram 23 alterações de aminoácidos nas subunidades A (18) e B (5), as quais geraram 16 variantes de LT. Entre estes variantes de LT, um mostrou efeito tóxico reduzido em comparação à toxina de referência LT1. A forma de LT atenuada (LT4) tem atividade enzimática reduzida devido à troca de aminoácido. / Enterotoxigenic Escherichia coli (ETEC) strains represent an important etiological agent of diarrheal disease, particularly among children and travelers in developing countries. Among the virulence factors expressed by ETEC strains the heat-labile (LT) and heat-stable (ST) enterotoxins represent the most revelevant phenotypes. Indirect evidences suggest that the severity of diarrhea associated to ETEC strains might reflect the natural diversity of wild strains to produce enterotoxins and/or the occurrence of variants endowed with reduced toxic effects. In the present study, we investigated both the genetic diversity of the etx operon, encoding the heat-labile toxin, and the capability to produce/secrete LT by ETEC strains isolated from humans or porcine in different geoghrafic areas. The results showed a remarkable variability on the production of LT with values ranging from 2 to 2,525 ng of toxin per ml of culture. LT secretion was also variable with values ranging from less than 0.04% to 49.5% of total LT produced by the different ETEC strains. Additionally, rabbit ileal loop experiments showed a good correlation between the amounts of secreted LT under in vitro conditions and fluid accumulation in vivo. We determined also the diversity of the etxAB operon of 50 ETEC strains (LT+ or LT+/ST+) belonging to different serotypes with emphasis to LT+-only producing strains isolated from asymptomatic children. The complete nucleotide sequences of the etxAB genes revealed 23 amino acid changes at the A (18) or B (5) subunits, which generated 16 variant forms of LT. Among these LT variants, one of them showed reduced toxic effects in comparison to the reference toxin LT1. The attenuated LT form (LT4) had decreased enzymatic activity due to an amino acid replacement (K4R) at the A1 subunit. LT4 retains its immunogenic and adjuvant properties following nasal immunization. Additionaly, the LT4 variant showed altered immune modulatory features and promoted a more biased Th1 response, which favor activation of effector CD8+ T lymphocytes, to co-administred antigen with regard to LT1. Taken together, our results demonstrate that ETEC strains isolated from human subjects express natural genetic variability leading to a remarkable polymorphism of the etx operon as well as production and secretion of LT. Such natural genetic diversity observed in ETEC strains may affect the host-pathogen relationships and, consequently, contribute to the severity of the disease among infected subjects.

A cárie dental é uma doença bacteriana infecciosa considerada como um dos principais problemas de saúde pública. O principal agente etiológico para o desenvolvimento da doença é o Streptococcus mutans. A proteína P1, também conhecida como antígeno I/II, do S. mutans é fundamental para a etapa inicial de adesão à superfície dental (sacarose-independente), sendo, portanto, considerada essencial para o processo de colonização deste patógeno. Algumas regiões dessa proteína vêm sendo empregadas como antígenos em estratégias vacinais contra a cárie, entre elas a região A, localizada na porção N-terminal, conhecida como região de ligação à saliva Saliva Binding Region (SBR). No entanto, apesar dos avanços, não existe vacina para a prevenção da cárie licenciada para uso em humanos. Diante disso, o presente trabalho tem como objetivo o desenvolvimento e caracterização de uma nova estratégia vacinal contra o S. mutans baseada em esquema de dose-reforço heterólogo, utilizando o fragmento P139-512, na forma de proteína purificada, expressa a partir de linhagens recombinantes de Bacillus subtilis. A proteína P139-512 compreende os aminoácidos 39-512 da proteína nativa, o que corresponde a toda região A e uma pequena porção da região variável da proteína P1. Utilizamos esporos de B. subtilis 1012, modificados para expressar o antígeno P139-512 (LDV702). A linhagem LDV704, além de expressar o antígeno, foi modificada para expressar uma invasina (InvA) com capacidade de se ligar a epitélios de mucosa. Camundongos da linhagem BALB/c foram imunizados por via sublingual com uma dose de esporos de B. subtilis (linhagens 1012, LDV702, LDV704 ou PBS) seguidos por dois reforços com a proteína P139-512, associada ou não com o adjuvante LTK63. Níveis significativos de anticorpos séricos foram induzidos pelas formulações em associação com o adjuvante após a terceira dose, e mostraram-se capazes de reconhecer os epítopos em diferentes linhagens de S. mutans. No entanto, nenhuma das formulações mostrou-se capaz de ativar respostas de mucosa (S-IgA). Porém, observamos que o adjuvante LTK63 empregado na estratégia dose-reforço heterólogo potencializou a resposta sérica de anticorpos IgG, sendo capaz de modular e melhorar qualitativamente as respostas induzidas. Assim, a administração das formulações na presença do adjuvante representa uma alternativa promissora para o controle do S. mutans. / Dental caries is an infectious bacterial disease considered as one of the main public health problems. The main etiological agent for the development of the disease is the Streptococcus mutans. The P1 protein, also known as S. mutans Ag I / II antigen, is essential for the initial stages of adhesion to the dental surface (sucrose-independent) and is, therefore, considered essential for the colonization process of this pathogen. Some regions of this protein have been used as antigens in vaccine strategies against caries, among them the A region, located at the amino terminal region also known as the Saliva Binding Region (SBR). However, despite the advances, there is no licensed anti-caries vaccine for human use. Therefore, the present work aims to develop and characterize a new vaccine strategy against S. mutans based on a heterologous priming/boost immunization regimen using the recombinant P139-512 fragment, expressed and purified from a Bacillus subtilis strain. The P139-512 protein comprises amino acids that encompasses the entire A region and a small portion of the variable region of the P1 protein. We used spores of B. subtilis 1012 (wild-strain) and recombinants that were modified to express the antigen P139-512 (LDV702). In addition to express the antigen, the LDV704 strain was modified to express a surface-exposed bacterial invasin (InvA) capable of binding to the mucosal epithelia. BALB/c mice were primed via the sublingual route with a dose of B. subtilis spores (1012, LDV702, or LDV704 strains) followed by two boosting doses with the purified protein P139-512, associated or not with the LTK63 adjuvant, by the same administration route. Significant serum antibody levels were induced by the formulations with the adjuvants after the third dose and the antibodies were shown to recognize epitopes exposed on the surface of different S. mutans strains. However, none of the formulations were capable to activate mucosal responses (S-IgA). Nevertheless, we observed that the LTK63 enhanced the serum IgG responses and qualitatively improved the induced antibody response. Thus, the administration of the formulations in the presence of the adjuvant represents a promising alternative for the control of S. mutans.

Chitosan is a biocompatible and biodegradable mucoadhesive polymer with unique advantages, such as the distinct trait of opening the junctions to allow paracellular transport of antigen and good tolerability. However, the poor solubility of chitosan in neutral or alkalinized media has restricted its applications in the pharmaceutical field. Chitosan can be easily carboxymethylated to improve its solubility in aqueous media, while its biodegradability and biocompatibility are preserved. Apart from this, carboxymethyl chitosan (CMCS) can be easily processed into nanoparticles which highlight its suitability and extensive usage for preparing different drug delivery formulations. The present study deals with the development and characterization of a delivery system based on CMCS nanoparticles using ovalbumin as model protein. We demonstrated that ovalbumin loaded nanoparticles were successfully synthetized using calcium chloride as a cross-linker by ionic gelation. The nanoparticles exhibited an average size of approximately 169 nm and presented a pseudo-spherical shape. The nanoparticles size increased according to the addition of CaCl2 due to the strong electrostatic attraction. During storage the nanoparticles size increased was attributed to swelling and aggregation. The loading efficiency of ovalbumin was found to be 17%. Confocal microscopy clearly showed the association between ovalbumin and CMCS chains into nanoparticles. Therefore, we suggest these nanoparticles can be considered as an attractive and promising carrier candidate for proteins and antigens. The major challenge that limits the use of such carriers is their instability in an aqueous medium. Thus, the next step of this work was to determine the robustness of several formulations using distinct freeze-drying protocols. This study demonstrated that mannitol in concentration of 10% (w/v) is well suited to preserve ovalbumin loaded CMCS nanocapsules from aggregation during lyophilization and subsequent reconstitution. Importantly, the results showed that an annealing step has a huge impact on porosity of freeze-dried cake by nearly complete crystallization of mannitol, once the crystalline matrix prevents the partial collapse and the formation of larger pores observed without annealing. Therefore, the usual observation that annealing increases the pore size due to growth of ice crystal size does not always apply, at least when crystallization of solute is involved. Since all characterizations and stability studies had been performed, the main purpose of this study was to develop a stable antigen delivery system for oral immunization using CMCS and inactivated rabies virus (RV) as the antigen. RV loaded nanoparticles was found to enhance both systemic (IgG) and local (IgA) immune responses against RV after oral delivery in mice. The effective doses 50% were 50-times higher than the negative controls, indicating that the immune response started only after the third boosting dose. Furthermore, enough neutralizing antibodies was produced to be protected against the harmful effects of the rabies virus. It is therefore concluded, that the CMCS nanoparticles formulated in this study, are suitable for oral vaccine delivery, and can be suggested as a promising delivery system for a diverse range of antigens as well as a gene/protein delivery system, especially for those positively charged. Since several approaches show that effective intervention in airway allergic inflammation can be achieved with allergen-activated interleukin-10-secreting cells, the final part of this work was dedicated to assessing whether IL-10 loaded chitosan nanoparticles (IL10-CSNPs) could be used as a possible inhalable therapeutic tool for preventing exacerbations in asthmatic patients. As positive controls, we also assess whether interleukin 17A and interleukin 9 have the ability to stimulate human airway smooth muscle (HASM) cell contractility using magnetic twisting cytometry (MTC). Significant decreased baseline cell stiffness was observed in HASM cells pre-treated with IL-10, but not with IL10-CSNPs, whereas treatment with IL-17A significantly enhanced baseline cell stiffening. Our findings reveal a previously unknown mechanism underlying immunotherapy for prevention and treatment of asthma. / A quitosana é um polímero mucoadesivo biocompatível e biodegradável, com vantagens únicas, tais como a característica distinta de abrir as junções que permitim o transporte paracelular de antígenos e boa tolerabilidade. No entanto, sua baixa solubilidade em meios neutros ou alcalinizados tem restringido suas aplicações no campo farmacêutico. A quitosana pode ser facilmente carboximetilada para melhorar de sua solubilidade em meios aquosos, enquanto sua biodegradabilidade e biocompatibilidade são preservadas. Além disso, a carboximetilquitosana (CMCS) pode ser facilmente processada na forma de nanopartículas, o que destaca sua adequabilidade para uso extensivo no preparo de sistemas de delivery de medicamentos. O presente estudo trata do desenvolvimento e caracterização de um sistema de delivery baseado em nanopartículas de CMCS utilizando ovalbumina como proteína modelo. Nós demonstramos que as nanopartículas carregadas com ovalbumina foram sintetizadas com sucesso utilizando cloreto de cálcio como agente de reticulação por gelificação iônica. As nanopartículas exibiram um tamanho médio de aproximadamente 169 nm e apresentaram uma forma pseudo-esférica. O tamanho das nanopartículas aumentou de acordo com a adição de CaCl2 devido à forte atração eletrostática. Durante o armazenamento, o tamanho aumentado das nanopartículas foi atribuído a incorporação de água e agregação. A eficiência de encapsulamento da ovalbumina foi de aproximadamente 17%. A microscopia confocal mostrou claramente a associação entre ovalbumina e a cadeias de CMCS nas nanopartículas. Sugerimos, portanto, que tal sistema pode ser considerado como candidato atraente e promissor para o carreamento de proteínas e antígenos. O principal desafio que limita o uso desses carreadores consiste na instabilidade em meio aquoso. Assim, o próximo passo deste trabalho foi determinar a robustez de várias formulações utilizandose diferentes protocolos de liofilização. Este estudo demonstrou que o manitol em uma concentração de 10% (p/v) é adequado para preservar da agregação as nanocápsulas de CMCS carregadas com ovalbumina durante a liofilização e subsequente reconstituição. Mais importante, os resultados mostraram que uma etapa de annealing tem um enorme impacto sobre a porosidade da amostra liofilizada devido a quase completa cristalização do manitol, uma vez que a matriz cristalina evita o colapso parcial e a formação de poros maiores observados na ausência do annealing. Portanto, a observação comum de que o annealing aumenta o tamanho doporos devido ao crescimento dos cristais de gelo nem sempre se aplica, pelo menos quando a cristalização de um soluto está envolvida. Uma vez que todas as caracterizações e estudos de estabilidade foram realizados, o principal objetivo deste estudo foi desenvolver um sistema estável de delivery de antígeno para imunização oral utilizando CMCS e vírus rábico inativado (RV) como antígeno. Verificou-se que as nanopartículas carregadas com RV aumentam as respostas imune sistêmica (IgG) e local (IgA) contra o RV após administração oral em camundongos. As doses efetivas 50% foram 50 vezes maiores que os controles negativos, indicando que a resposta imune foi iniciada apenas após a terceira dose da vacina. Além disso, foram produzidos anticorpos neutralizantes suficientes para proteção contra os efeitos nocivos do vírus rábico. Conclui-se, portanto, que as nanopartículas de CMCS formuladas neste estudo, são adequadas para o delivery oral de vacinas, e podem ser sugeridas como um sistema promissor de delivery para uma gama diversa de antígenos, bem como para o delivery de genes/proteínas, especialmente para aqueles carregados positivamente. Uma vez que diversas abordagens mostram que uma intervenção efetiva em casos de inflamação alérgica de vias aéreas pode ser conseguida por meio de células secretoras de interleucina 10 (IL-10) mediante ativação por alergenos, a parte final deste trabalho esteve dedicada a avaliação de nanopartículas de quitosana carregadas com IL-10 (IL10-CSNPs) como possível ferramenta terapêutica inalável para prevenção de exacerbações em pacientes asmáticos. Como controles positivos, avaliou-se adicionalmente se as interleucinas 17A (IL-17A) e 9 (IL-9) possuem a capacidade de estimular a contratilidade de células humanas de músculo liso de vias aéreas humanas (HASM) por meio de citometria de torção magnética (MTC). Uma diminuição significativa da rigidez celular basal foi observada em células HASM pré-tratadas com IL-10, mas não com IL10-CSNPs, enquanto que o tratamento com IL-17A aumentou significativamente a magnitude rigidez celular basal. Nossos resultados revelam um mecanismo previamente desconhecido subjacente à imunoterapia para prevenção e tratamento da asma.