Dendritic cells (DCs) are prime mediators of innate and adaptive immunity. In humans the DC population comprise only 0.1% of all leukocytes, making their isolation and ex vivo manipulation difficult. Since study of DC activity in vitro requires large numbers of DCs to be readily available, a cell line model, KG-1, was selected. KG-1 cells are a cytokine-responsive human CD34+ myelomonocytic cell line and can be induced to differentiate to a DC phenotype. A range of differentiation agents and protocols were compared, and differentiation efficiency was determined using both morphological features and cell surface marker expression. Expression of CD83, CD11c, CD123, CD86, HLA-DR and DC-SIGN was assessed by immunofluorescence and flow cytometry. KG-1 cells stimulated with 10 ng/ml PMA and 100 ng/ml Ionomycin were found to be the ideal model for obtaining Dendritic Like Cells (DLCs) in vitro. The effect of lactic acid bacteria on KG-1 differentiation was also tested using two immunomodulatory strains, Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052. After 5 days of incubation with R0011 the KG-1 cells expressed DC-specific surface markers CD83, CD86, CD11c, CD123, DC-SIGN and HLA-DR. Lactobacillus rhamnosus R0011 induced a marked rise in CD83 expression with a mean fluorescence intensity of 115.3 after 5 days, suggesting this strain promoted KG-1 differentiation to DLC. Analysis of cytokine by KG-1 DLC indicated that constitutive production of pro-inflammatory cytokines TNF-α and IL-12 was minimal. However IL-10 and TGF-β were detected after TLR-agonist stimulation of R0011-differentiated KG-1s. This study aimed to develop and assess the KG-1 cell model for screening effects of mediators and microbes on DC. / UOIT

The ecological niche of the vagina contains a large number of different microbes that are constantly interacting with each other and the host. Culture methods have not been sufficient in order to resolve the complexity of the normal vaginal flora. Further, the methods for delineating normal flora from not normal flora are not easily handled and are traditionally not based on culture but on microscopy of elements of the vaginal fluid. In the work presented in this thesis, an international collaboration was established that pin-pointed some of the difficulties in classifying vaginal floras, including staining, sampling, and discordance when lactobacilli are few in number, and that emphasized the importance of the size of the vision field in microscopes. As lactobacilli are prominent members of the normal vaginal flora they need to be carefully classified if further work towards more robust scoring tools is to be achieved. Phenotypic methods have not been able to separate the closely related Lactobacillus species of the vagina. Progress in molecular biology has provided possibilities to characterize these lactobacilli, which are mainly from the Lactobacillus acidophilus group. In this work a large number of strains collected by true random sampling were subjected to RAPD-PCR, TTGE and multiplex PCR for species identification. The major species found were L. crispatus, L. gasseri and L. jensenii and the recently described L. iners. The presence of L. iners has not been detected in previous studies due to its special nutrient requirements. Development of pyrosequencing technology also made it possible to match signatures of the two variable regions V1 and V3 of the 16S rRNA gene of the vaginal lactobacilli and identify them to the species level in a high throughput manner. The study confirmed that the dominating flora in women with normal vaginal flora comprises the four species mentioned previously. Repetitive sampling during IVF-treatment with highly varying oestrogen levels demonstrates changes that possibly occur during changes in the natural life cycle. Furthermore, L. iners was found to be the first species to be established after spontaneously resolved or treated Bacterial Vaginosis. These findings can be of help in developing new strategies for regaining and retaining the normal vaginal flora.

In a cross-sectional study the extent and distribution of dental caries was studied in 817 children, 4, 8 and 13 years old in three areas inSweden. The importance of dietary habits, sugar intake, social conditions, professional dental care, oral hygiene and the use of fluorides on caries prevalence was analyzed. In a subsample, con­sisting of 88 8-year-olds and 91 13-year-olds the net caries increment during aone-year period was studied.In this longitudinal study, the salivary levels of lactobacllli and S. mutans were determined and correlated to sugar intake and caries increment. The cross-sectional study showed obvious differences in caries preva­lence among the studied areas. These differences could mainly be explained by variations in the age from which children had received professional dental care, frequency of tooth-brushing and the use of topical fluorides. There were no differences in dietary habits or sugar intake that could explain the differences in caries prevalence among the areas studied. Examples are given of how changes in some determinants may affect the caries prevalence by means of path analysis. The longitudinal study showed that caries Increment was higher when sugar intake and meal frequency were high (&gt; the mean value for the age group). The highest mean caries increment was found in children with high prevalence of lactobacllli and S. mutans in the saliva combined with a high sugar intake. Groups of children with high salivary levels of both Iactobac ì II i and S. mutans developed 3-4 times more caries than other children. This relation did not always exist in the Individual case. Children with a low caries Increment during one year (0-2 surfaces) brushed their teeth more often and rinsed their mouths more frequently with fluoride solution than children with a high caries increment (&gt; 3 surfaces). The use of fluoridated toothpaste was somewhat more common, however, in the low carles increment group only in the.8-year-oIds. The gingival status was used as a measure of oral hygiene and gingivitis scores revealed statistically significant differences between groups with a low caries increment and a high carles increment (p &lt; 0.01, p &lt; 0.05) in the two age groups, respectively. With the variables frequency of meals, total sugar intake, salivary level of lactobacilli and S. mutans, oral hygiene and use of topical fluorides the net caries increment during one year could be correctly predicted in 79 % of the 8-year-olds and 81 % of the 13-year-olds when the children were divided Into two groups according to their net car­ies increment; 0-2 surfaces and &gt; 3 surfaces. / <p>S. 1-43: sammanfattning, s. 45-116: 5 uppsatser</p> / digitalisering@umu

Le microbiote intestinal affecte la plupart des processus physiologiques de l’hôte, et plus particulièrement la digestion et le métabolisme. Cependant, les mécanismes moléculaires en jeu restent encore peu connus. Pour répondre à cette question, nous utilisons un modèle gnotobiotique simple : la larve de Drosophile monoassociée à un de ces symbiont naturel : Lactobacillus plantarum. Notre groupe a montré que L. plantarum promeut la croissance larvaire d’individus soumis à une carence en protéines. En effet, les larves monoassociées à L. plantarum se développent bien plus rapidement que des individus axéniques. Ainsi, L. plantarum tempère l’effet délétère de la carence nutritionnelle. Cette amélioration de la croissance repose en partie sur la hausse du niveau d’expression des protéases digestives de l’hôte ainsi que sur la modulation de la voie de signalisation TOR (Target Of Rapamycin) de la Drosophile par les bactéries symbiotiques. Notre travail s’est focalisé sur la recherche d’autres mécanismes génétiques impliqués dans l’interaction entre la Drosophile et L. plantarum au cours de la croissance larvaire. Nos résultats montrent que les variations génomiques naturelles de la Drosophile affectent l’intensité du bénéfice de croissance conféré par L. plantarum. En outre, les bases de notre étude ont permis de mettre en évidence que le microbiote intestinal a la capacité d'agir comme "tampon génétique" en compensant les défauts de croissance causés par le fond génétique des mouches. De plus, L. plantarum permet de diminuer la variation phénotypique de plusieurs caractères de la mouche tels que la croissance, la taille de certains organes et la durée du cycle larvaire. Nous avons également identifié le gène dawdle, codant un ligand de la voie TGF-β, comme acteur de l’interaction Drosophile-L. plantarum. De plus nous avons montré que Dawdle régule les protéases digestives de la Drosophile dans un contexte nutritionnel de carence en protéine, et cette régulation peut-être activatrice ou bien inhibitrice selon l’environnement microbien. / Intestinal microbiota can modulate virtually all aspects of their host physiology, and particularly, digestion and metabolism. However, the molecular mechanisms at play remain largely unknown. To tackle this question, we use a simple gnotobiotic model: Drosophila larvae monoassociated with one of its major natural symbiont, Lactobacillus plantarum. Previous work from our group showed that L. plantarum promotes the juvenile growth of larvae facing a protein scarcity, thereby dampening the deleterious effect of the nutrient deficiency on larval growth. This growth enhancement partially relies on the upregulation of intestinal proteases, as well as on the modulation of the host TOR (Target Of Rapamycin) pathway by the symbionts. My thesis work aimed at unraveling other host genetic mechanisms involved in the interaction between Drosophila and L. plantarum during growth. Our work showed that host natural genomic variations affect the fly physiologic response to L. plantarum. Furthermore, the bases of our work enabled to unveil a novel role of intestinal bacteria, revealing their ability to act as a genetic buffer to compensate the growth impairments due to the fly genetic background. In addition, L. plantarum decreases the phenotypic variations in various host fitness traits (growth, organ size, timing to pupariation) and it also confers robustness to organ patterning. Finally, we showed that the TGF-β ligand, Dawdle plays an important regulatory role on digestive enzymes in a protein-deficient nutritional context, and that this regulation can be inhibitory or activating depending on the microbial environment.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-01-30T12:24:19Z No. of bitstreams: 1 arquivo total.pdf: 605461 bytes, checksum: e147ffe79147860c37a2b571b23b9cbc (MD5) / Made available in DSpace on 2017-01-30T12:24:20Z (GMT). No. of bitstreams: 1 arquivo total.pdf: 605461 bytes, checksum: e147ffe79147860c37a2b571b23b9cbc (MD5) Previous issue date: 2016-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The cashew (Anacardium occidentale L.) is a tropical, popular and well accepted fruit, due its nutritional characteristics and sensory attributes. The processing of the cashew pseudo-fruit (also called peduncle) generates the main byproduct which represent up to 20% of the total weight of the pseudo-fruit. In the last years, studies verifying the prebiotic potential of the byproducts from the processing of the cashew peduncle are scarce or nonexistent. In this context, the present study aimed to evaluate in vitro the prebiotic effects of a lyophilized powder obtained from the byproduct of the cashew peduncle (named CAP) against different probiotic strains of Lactobacillus, such as: L. acidophillus LA-05, L. casei L-26 and L. paracasei L-10. The growth and viable cell numbers of the Lactobacillus strains were monitored in Mann, Rogosa and Sharpe (MRS) broth containing CAP (20 g/L), glucose (20 g/L) and fructooligosaccharides – FOS (20 g/L), during 48 h. Some parameters related to the metabolic activity of the probiotic strains, cultivated in these different culture media, were evaluated, such as pH, production of organic acids and consumption of sugars. Similar viable cell count for each tested Lactobacillus strain in the different culture media were observed, count reached 8.5 - 9.0 log CFU/mL and pH values at approximately 4.0 after 48 h of cultivation. The growth of these Lactobacillus strains in MRS broth containing glucose, FOS or CAP resulted in a decrease of pH, production of organic acids (acetic, citric, malic, formic and lactic) and consumption of sugars (glucose, fructose and maltose) during the incubation time, indicating an intense bacterial metabolic activity. Overall, the tested Lactobacillus strains showed similar capability to ferment the FOS and the CAP, although some differences related to the production of organic acids and consumption of sugars were detected. The results showed that the CAP possesses a prebiotic potential effect toward Lactobacillus strains. These findings may stimulate the agro-industrial sector to valorize the byproducts from the cashew pseudo-fruit processing as an added-value ingredient to the food industry. / O caju (Anacardium occidentale L.) é uma fruta tropical, amplamente difundida e valorizada, tanto por suas características nutricionais quanto pelos seus atributos sensoriais. O principal subproduto gerado do caju decorre do processamento do seu pedúnculo (pseudofruto), representando até 20% do peso total do pseudofruto. Com base nesse contexto, esse estudo teve por objetivo avaliar in vitro os efeitos prebióticos de um pó liofilizado obtido a partir do subproduto do pedúnculo do caju (denominado CAP) frente a diferentes cepas probióticas de Lactobacillus, a citar: L. acidophillus LA-05, L. casei L-26 e L. paracasei L-10. O crescimento e o número de células viáveis das cepas de Lactobacillus foram monitorados em caldo de Mann, Rogosa e Sharpe (MRS), contendo o CAP (20 g/L), glicose (20 g/L) e frutooligossacarídeos - FOS (20 g/L), durante um período de 48 h. Também foram avaliados alguns parâmetros relacionados à atividade metabólica das cepas probióticas ensaiadas quando cultivadas nos diferentes meios de cultura, como os valores de pH, produção de ácidos orgânicos e o consumo de açúcares. Para cada cepa de Lactobacillus ensaiada, foram observadas contagem similares de células viáveis nos diferentes meios de cultivo, sendo alcançadas contagens entre 8.5 – 9.0 log UFC/mL e valores de pH em torno de 4.0, ao final do cultivo de 48 h. O cultivo de todas as cepas de lactobacilos em MRS contendo glicose, FOS ou CAP resultou em decréscimos de pH, produção de ácidos orgânicos (acético, cítrico, málico, fórmico e lático) e consumo de açúcares (glicose, frutose e maltose) ao longo do tempo de incubação avaliado, indicando intensa atividade metabólica bacteriana. Em geral, todas as cepas de lactobacilos testadas apresentaram capacidade semelhante para fermentar FOS e CAP, embora algumas diferenças quantitativas relacionadas com a produção de ácidos orgânicos e consumo de açúcares tenham sido detectadas. Os resultados deste estudo mostraram que o CAP possui potencial prebiótico frente a cepas de Lactobacillus. Estes resultados podem estimular o setor agro-industrial para valorizar os subprodutos do processamento do pendúnculo do caju como um ingrediente com valor agregado para a indústria de alimentos.

Le peptidoglycane (PG) est le composé majeur de la paroi des bactéries à Gram positif. Il est constitué de chaines de sucres, formées de l’alternance de N-acétyl-glucosamine (GlcNAc) et d’acide N-acétyl-muramique (MurNAc) et reliées entre elles par des chaines peptidiques. Cette structure confère à la bactérie une grande résistance mais aussi une certaine flexibilité qui lui permettent de grandir et de se diviser tout en gardant sa forme. Cette dualité entre rigidité et flexibilité est assurée par un équilibre entre l’activité des enzymes qui polymérisent le PG, les protéines liant la pénicilline (PBP), et de celles qui l’hydrolysent, les hydrolases du PG (PGH). Pendant ou après sa synthèse, la structure du PG peut subir différentes modifications, qui vont moduler l’activité des enzymes de synthèse et dégradation du PG. Au cours de ce travail, nous avons caractérisé les modifications structurales du PG chez deux espèces de lactobacilles et étudié leur rôle fonctionnel. Nous avons identifié la première amidotransférase responsable de l’amidation de l’acide méso-diaminopimélique et montré l’influence de cette modification sur l’activité d’une PGH, la L,D-carboxypeptidase DacB, ainsi que sur la synthèse du PG septal par les PBPs chez Lactobacillus plantarum. Nous avons ensuite mis en évidence pour la première fois une O-acétylation des GlcNAc en plus de l’O-acétylation des MurNAc, ces deux modifications étant réalisées par deux O-acétyl-transférases distinctes, OatA et OatB, qui jouent des rôles antagonistes dans le contrôle de l’activité des PGHs chez L. plantarum. Nous avons aussi révélé l’implication de l’O-acétyl-transférase OatA dans le contrôle de la septation. Enfin, nous avons montré l’influence de l’O-acétylation des MurNAc du PG sur les propriétés anti-inflammatoires d’une souche de Lactobacillus casei. / Peptidoglycan (PG) is the major component of the gram positive cell wall. It is composed of glycan chains formed by the polymerization of the N-acetylglucosamine-N-acetyl muramic acid heterodimer, and cross-linked by peptidic stem. This structure confers high resistance to the bacterial cell wall but also some flexibility allowing growth and shape maintenance. This duality between rigidity and flexibility is the result of a steady-state between the PG polymerizing enzymes, the penicillin binding protein (PBP) and the PG hydrolases (PGH). More or less concomitantly with its synthesis, certain modifications can occur on PG structure that will modulate the activity of PG synthesis and degradation enzymes.During this work, we have characterized the PG structural modifications in two lactobacilli species and studied their functional role. We have identified the first amidotransferase involved in meso-diaminopimelic acid amidation and shown the influence of this modification on the activity of the L,D-carboxypeptidase, DacB, and also on the septal PG synthesis by the PBP in Lactobacillus plantarum. Then, we have highlighted for the first time, the presence of O-acetylation on GlcNAc in addition to O-acetylation on MurNAc. These two modifications are catalyzed by two dedicated O-acetyltransferases, OatB and OatA respectively, that control PGH activity in an antagonistic way. We have also demonstrated the implication of the OatA O-acetyltransferase in septation control. Finally, we have shown the influence of PG MurNAc O-acetylation on the anti-inflammatory properties of a Lactobacillus casei strain.

At birth, the immune system is immature and the gut microbiota influences immune maturation. Staphylococcus aureus (S. aureus) and lactobacilli are part of the neonatal gut microbiota and have seemingly opposite effects on the immune system. S. aureus is a potent immune activator and early-life colonization associates with higher immune responsiveness later in life. Lactobacilli-colonization associates with reduced allergy-risk and lower immune responsiveness. Further, lactobacilli modulate immune-activation and have probiotic features. Here, we investigated S. aureus-induced activation of human lymphocytes, including T regulatory cells (Tregs), conventional T-cells (CD4+ and CD8+), unconventional T-cells (γδ T-cells and MAIT-cells) and NK-cells from children and adults, together with the modulatory effect of lactobacilli on immune-activation. Further, early-life colonization with these bacteria was related to lymphocyte-maturation, plasma cytokine- and chemokine-levels and allergy.  S. aureus cell free supernatant (CFS) and staphylococcal enterotoxin (SE) A induced an increased percentage of FOXP3+ Tregs and of CD161+, IL-10+, IFN-γ+ and IL-17A+ Tregs (Paper I). The same pattern was observed in children with a lower degree of activation, possibly due to lower CD161-expression and poor activation of naive T-cells (Paper II). S. aureus-CFS induced IFN-γ-expression, proliferation and cytotoxic capacity in conventional and unconventional T-cells, and NK-cells. SEA, but not SEH, induced activation of unconventional T-cells and NK-cells by unknown mechanism(s) (Paper III, extended data). Lactobacilli-CFS reduced S. aureus-induced lymphocyte activation without the involvement of IL-10, Tregs or monocytes, but possibly involving lactate (Paper III). Early-life colonization with S. aureus associated with increased percentages of CD161+ and IL-10+ Tregs while lactobacilli-colonization negatively correlated with the percentage of IL-10+ Tregs later in life (Paper II). Allergic disease in childhood associated with double allergic heredity, being born wintertime and with higher plasma levels of TH2-, TH17- and TFH-related chemokines early in life. Lactobacilli-colonization associated with lower prevalence of allergy, reduced chemokine-levels and increased levels of IFN-γ in plasma (Paper IV).    This thesis provides novel insights into S. aureus- and SE-mediated activation of Tregs, unconventional T-cells and NK-cells and suggests an overall impairment of immune-responsiveness towards this bacterium in children. Further, S. aureus-colonization may influence the maturation of peripheral Tregs. Our data show that lactobacilli potently dampen lymphocyte-activation in vitro and that colonization associates with Treg-responsiveness, altered plasma cytokine- and chemokine-levels and with remaining non-allergic, thereby supporting the idea of lactobacilli as important immune-modulators. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>

Giardia duodenalis est le protozoaire responsable de la giardiose, la parasitose intestinale la plus répandue dans le monde. Cette infection se caractérise par une malabsorption intestinale, des diarrhées, une perte de poids et des douleurs abdominales intenses chez l’Homme et de nombreux mammifères. Par ailleurs, cette maladie dont l’impact en santé publique et vétérinaire est reconnu, peut entraîner d’importantes déficiences nutritionnelles en particulier chez les sujets jeunes. L’infection est causée par l’ingestion de kystes de Giardia duodenalis (syn. G. lamblia, G. intestinalis) présents dans les aliments ou l’eau contaminée. Infectieux à très faibles doses, ces kystes survivent pendant plusieurs semaines dans l’environnement et sont résistants aux différents désinfectants. Suite au dékystement, la forme végétative de Giardia, le trophozoïte, adhère à l’épithélium intestinal au niveau des parties supérieures de l’intestin grêle et se multiplie, causant les symptômes. Cette phase se termine par un nouvel enkystement et l’excrétion de kystes par les fèces. Le nombre croissant d’infections liées à la contamination de l’eau potable, à l’émergence de souches résistantes aux médicaments disponibles, à la fréquence des échecs thérapeutiques et à l’importance des effets secondaires associés aux traitements font de cette maladie un sujet d’actualité de plus en plus préoccupant qui nécessite le développement de traitements alternatifs. Il est désormais bien établi que le microbiote et/ou certaines souches de bactéries probiotiques ont un impact bénéfique dans la giardiose. En particulier, la bactérie probiotique Lactobacillus johnsonii La1 (LjLa1) a un rôle protecteur contre la croissance de Giardia in vitro et in vivo. Nous avons cherché dans ce travail de Thèse à décrypter les mécanismes moléculaires associés à l’effet inhibiteur des facteurs sécrétés par LjLa1. Nous avons montré qu’in vitro, LjLa1 agissait en libérant des enzymes de type Bile Salt Hydrolases (BSH) qui modifient alors des composants de la bile non-toxiques pour le parasite (sels biliaires conjugués) en des composants toxiques (sels biliaires déconjugués). Les 3 gènes BSH codés dans le génome de LjLa1 ont été clonés chez Escherichia coli et les protéines taguées histidine purifiées pour étudier leurs propriétés biochimiques et biologiques. Obtenues sous forme active, nous avons pu en définir les spécificités de substrats et montrer qu’elles sont capables d’inhiber significativement la croissance de G. duodenalis in vitro et in vivo, dans un modèle murin de la giardiose (souriceaux OF1 non sevrés). En parallèle, nous avons identifié, à l’issue d’un large criblage de souches de lactobacilles selon leur activité anti-Giardia in vitro, une souche probiotique aux effets inhibiteurs comparables à ceux de LjLa1 : Lactobacillus gasseri CNCM-4884. Administrée in vivo dans le modèle murin de la giardiose, cette souche a réduit de 93% la charge parasitaire dans l'intestin grêle des nouveaux nés et a également réduit de façon significative le nombre de kystes libérés dans l’environnement, permettant ainsi de réduire la transmission de Giardia. Des travaux parallèles ont été réalisés au cours de ce projet de Thèse, notamment le développement d’outils de moléculaire pour l’expression hétérologue de molécules d’intérêt en santé animale chez divers lactobacilles. Le développement de ces « vecteurs mucosaux » permettra à terme de proposer une stratégie de surexpression de BSH par les lactobacilles afin d’accroitre l’activité BSH in vivo, et renforcer ainsi l’élimination du parasite. Ces résultats permettent de proposer de nouvelles pistes thérapeutiques originales contre les giardioses humaines et animales, basées sur l’utilisation de lactobacilles probiotiques ou sur les activités BSH qui en sont dérivées. Ces traitements offrent alors une alternative sérieuse aux antibiotiques et permettront de pallier aux actuels fréquents échecs thérapeutiques. / Giardia duodenalis is a protozoan parasite responsible for giardiasis, the most common intestinal parasitic disease worldwide. This infection is characterized by intestinal malabsorption, diarrhea, weight loss and abdominal pain in humans and various mammalian species. Besides, this disease has a high veterinary and public health impact, leading to important nutritional deficiencies in young subjects. The infection is caused by the ingestion of food or water contaminated with infectious cysts of the parasite. Giardia cysts can survive for several weeks in the environment and are highly resistant to disinfectants. Giardia excysts in the intestinal tracts of its host and replicates under the trophozoite stage causing the symptoms. Trophozoites adhere to the intestinal epithelium of the small intestine and multiply, causing the symptoms. The cycle ends by a new encystment and infectious cysts are released in environments with feces. The increasing number of giardiasis cases, mainly due to water contaminations, the emergence of parasite strains resistant to drugs and therapeutic failures, make research on alternative therapeutic strategies and treatments highly needed. Nowadays, it is well known that the microbiota and probiotics play an important role in protection against this parasite. For instance, the probiotic strain Lactobacillus johnsonii La1 (LjLa1) prevents the establishment of Giardia in vitro and in vivo. In this thesis, we have tried to point out the molecular mechanism(s) involved in this inhibitory effect(s). We showed in vitro that LjLa1 was releasing "Bile Salt Hydrolases" (BSH) – like activities that modify some components of bile (conjugated bile salts) into toxic compounds (deconjugated bile salts) for Giardia. We have cloned and expressed each of the three bsh genes present in the genome of LjLa1 in Escherichia coli in order to study their enzymatic and biological properties. Two BSH were obtained as recombinant active enzymes and biochemical tests showed that they have distinct substrate specificities despite similar predicted 3D structures. Moreover, these two BSHs of LjLa1 exhibited anti-giardial effects in vitro and in vivo in a murine model of the giardiasis (OF1suckling mice), comforting the hypothesis of the biological role of active BSH, derived from probiotics, against Giardia. A wide collection of diverse lactobacilli strains was screened to assess their effectiveness to also display both anti-giardial and BSH activities. This screening allowed the identification of several strains exhibiting strong anti-giardial effects such as Lactobacillus gasseri CNCM I-4884. In a murine model of giardiasis, this strain dramatically reduced the parasite burden in the small intestine of treated animals and significantly reduced the number of cysts in the colon, which could contribute to blockage of parasite transmission in environments. Additional studies were realized in parallel in order to explore the potency of lactobacilli to exert beneficial effects on health. For this, molecular tools were successfully developed in various lactobacilli strains to express and deliver therapeutic molecules at mucosal surfaces. The development of these tools will further allow the overexpression of BSH by lactobacilli to increase their in vivo BSH-activity and strengthen the elimination of the parasite. Altogether, this thesis work proposes new original therapeutic strategies against human and animal giardiasis, based on the use of BSH-positive lactobacilli strains or recombinant BSH- derived from probiotic strains, to counteract the frequent therapeutic failures, offering a serious alternative to antibiotics.

Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42–66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Abstract Dental caries in toddlers was studied in relation to several risk factors which were also determined by salivary tests in a longitudinal design. Another specific aim was to reveal the effect of prolonged pacifier sucking on caries development. The study population consisted of 183 pre-school children. At the baseline, salivary microbiological tests were taken from children whose average age was 2.5 years. Risk factors for colonisation of salivary lactobacilli and yeasts were determined from a questionnaire filled in by the parents. At the 2-year follow-up, caries in primary teeth was studied against the possible risk factors recorded at the baseline. At the 7-year follow-up, the risk factors identified at the age of two were analysed against caries development in primary molars and in first permanent molars. Also the long-term predictive value of salivary microbiological tests was investigated. At the baseline, the use of pacifier and nocturnal use of nursing bottle were associated with colonisation of salivary lactobacilli and yeasts. Prolonged pacifier sucking and use of nursing bottle at nights were shown to be associated with caries development in children at the 2-year follow-up. Consumption of sweets, lack of daily tooth brushing and nocturnal use of nursing bottle at the age of two were associated with caries onset in both primary and permanent molars at the seven-year follow-up. The use of fluoride tablets reduced the risk of caries onset in primary molars. Children who were colonised by salivary lactobacilli or yeasts at the baseline were susceptible to caries in primary molars. Early colonisation of lactobacilli was associated with caries in permanent molars. The results suggest that the risk of caries is possible to assess in toddlers by identifying caries-related habits early, already at the age of two. Microbiological tests may also have some value. Assessment of caries risk in toddlers enables both prevention and early intervention, and thereby prevention of caries development in children. / Tiivistelmä Tämän tutkimuksen tarkoituksena oli pitkäaikaisseurannassa selvittää taaperoikäisiltä lapsilta otettujen sylkitestien ja muiden määritettyjen riskitekijöiden mahdollista yhteyttä karieksen kehittymiseen. Erityisesti haluttiin myös tutkia pitkittyneen tutin käytön merkitystä. Tutkimusaineisto koostui 183:sta alle kouluikäisestä lapsesta. Aloitusvaiheessa otettiin mikrobiologiset sylkitestit lapsilta, joiden keski-ikä oli 2,5 vuotta, ja määritettiin anamnestisesti mahdolliset riskitekijät laktobasillien ja hiivasienten kolonisaatiolle. Kahden vuoden seurannan jälkeen tutkittiin riskin toteutumista maitohampaiden karioitumisessa. Seuraavassa seitsemän vuoden seurantatutkimuksessa tutkittiin karieskehitystä maitomolaareissa ja ensimmäisissä pysyvissä molaareissa kahden vuoden iässä määritetyissä riskiryhmissä. Myös syljen mikrobiologisten testien avulla määritetyn riskin toteutumista selvitettiin seitsemän vuoden seurannan päätyttyä. Tutkimuksen aloitusvaiheen sylkitestitulokset osoittivat, että tutin käyttö ja tuttipullon käyttö öisin olivat yhteydessä syljen kariekseen liittyviin mikrobeihin, ja ne voivat siten lisätä kariesaktiivisuutta maitohampaistossa. Kahden vuoden seurantatutkimuksessa todettiinkin, että karieskehitys oli yhteydessä aikaisemmin määritettyihin riskitekijöihin: pitkittyneeseen tutin käyttöön ja tuttipullon käyttöön öisin. Seitsemän vuoden seurantatutkimuksen tulokset osoittivat, että makeisten käyttö kaksivuotiaana, puutteellinen hampaiden harjaus ja tuttipullon käyttö öisin ovat riskitekijöitä sekä maitomolaarien että ensimmäisten pysyvien molaarien karieskehitykselle. Fluoritablettien käyttö vähensi maitomolaarien karioitumista. Myös varhain tehdyillä syljen mikrobiologisilla testeillä oli pitkäaikainen ennustusarvo. Tulokset osoittavat, että riskin määritys taaperoikäisten lasten karieskehitykselle voidaan tehdä varhaisessa vaiheessa sekä mikrobiologisten sylkitestien avulla että määrittelemällä muiden mahdollisten riskitekijöiden esiintyvyys. Varhaisen kariesriskin määrityksen avulla voidaan ehkäisevät hoitotoimenpiteet kohdistaa ajoissa riskiryhmille ja siten ehkäistä karieskehitystä ja korjaavan karieshoidon tarvetta lapsilla.