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The gastrointestinal mucosal immune system has the daunting task of maintaining immune homeostasis by eliminating potentially harmful microorganisms and limiting tissue injury while inducing tolerogenic responses to luminal antigens including innocuous food, commensal bacteria and self-antigens. This carefully orchestrated system depends on elaborate down-regulating mechanisms that mediate and maintain a state of tolerance under normal conditions. Changes in such delicate balance are linked to the development of gastrointestinal pathology as well as systemic disease states. Despite the rapid increase in our appreciation of the gastrointestinal immune system, there is still a major disconnect between the description of how mucosal immune responses are organized and controlled and an insufficient mechanistic understanding of how such responses shape and influence disease outcome and pathogenesis. By using model enteric microorganisms Helicobacter pylori and Clostridium difficile, this dissertation presents a systematic effort to generate novel mechanistic hypothesis based on computational predictions and experimentally elucidate the mechanisms of action underlying mucosal immune responses and pathology in the gut. In this thesis I present i) an overview on mucosal immunology and the need to develop novel therapeutics that limit the pathogenic effects of invading bacteria while maintaining their protective functions, ii) the role of miRNAs in the modulation of immune responses to enteric pathogens, iii) the mechanisms by which Helicobacter pylori is able to limit effector inflammatory responses required for bacterial clearance thus favoring tolerance over immunity, iv) intracellular mechanisms of immune evasion that contribute to bacterial persistence and chronic infection. The knowledge generated throughout this dissertation exemplifies how a combination of computational modeling, immunoinformatics and experimental immunology holds enormous potential for discovering unforeseen targets and developing novel vaccines and cures for infectious, allergic and immune-mediated diseases. / Ph. D.

The mucosal immune system encompasses a wide array of interactions that work in concert to protect an individual from harmful agents while retaining tolerance to molecules, microbes, and self-antigens that present no danger. The upheaval in the regulation-response balance is a critical aspect in both infectious and immune-mediated disease. To understand this balance and methods of its restoration, iterative and integrative modeling cycles on the pathogenesis of disease are necessary. In this thesis, I present three studies highlighting phases of a systems immunology cycle. Firstly, the thesis provides a description of the construction of a computational ordinary differential equation based model on the host-pathogen-microbiota interactions during Clostridium difficile infection and the use of this model for the development of the hypothesis that host-antimicrobial peptide production may correlate with increased disease severity and promote increased recurrence. Secondly, it provides insight into the necessity of trans-disciplinary analysis for the understanding of novel molecular targets in disease through the immunometabolic regulation of CD4+ T cell by NLRX1 in inflammatory bowel disease. Third, it provides the assessment of novel therapeutics in disease through the evaluation of LANCL2 activation in influenza virus infection. In total, the computational and experimental strategies used in this dissertation are critical foundational pieces in the framework of precision medicine initiatives that can assist in the diagnosis, understanding, and treatment of disease. / Ph. D.

The mucosal immunity of the gastrointestinal (GI) tract is constituted by a complex, highly specialized and dynamic system of immune components that aim to protect the gut from external threats. The sustained exposure of the mucosal immune system of the GI tract to an enormous number of lumen antigens, requires the constant upkeep of a highly regulated balance between initiation of immune responses against harmful agents and the generation of immune tolerance towards innocuous antigens. Therefore, the regulatory component is key to preserve tissue homeostasis and a normal functioning of the system. Indeed, defective regulatory responses lead to the development of pathological conditions, including unresolved infections, and inflammatory diseases. In this study, we aim to elucidate novel mechanisms involved in host-pathogen interactions during Helicobacter pylori and Clostridium difficile infections. Indeed, this work integrates preclinical in vivo and in vitro experimental approaches together with a bioinformatics pipeline to identify and characterize novel regulatory mechanisms and molecular targets of the mucosal immune system during enteric infections. Firstly, we identified a novel regulatory mechanism during H. pylori infection mediated by a specific subset of IL10-producing tissue resident macrophages. Secondly, we employed an ex vivo H. pylori co-culture with bone marrow derived macrophages, that together with a global transcriptomic analysis and a bioinformatics pipeline, lead to the discovery of promising regulatory genes based on expression kinetics. Lastly, we characterized the innate inflammatory responses induced during the course of C. difficile infection and identified IL-1ß, and its subsequent induction of neutrophil recruitment, as a key mediator of C. difficile-induced effectors responses. The characterized regulatory mechanisms in this work show promise to lead the generation of new host-centered therapeutics through the modulation of the immune response as promising alternative treatments for infectious diseases. / Doctor of Philosophy / The immune system is responsible for protecting the human body from external threats. To achieve this goal, it must differentiate between harmless and harmful agents to only fight the latter. To combat these dangerous agents, the immune system induces highly controlled, inflammatory processes that aim to eliminate the external threat while minimizing the damage of human tissues and organs. The gastrointestinal tract is exposed to an enormous number of molecules, mostly harmless molecules from both the ingested food and the beneficial bacteria inhabiting the gut, but also from harmful bacteria and agents, only separated from the internal body structures by a thin layer called the epithelial barrier of the gut. The immune system responsible for the protection of the gastrointestinal tract includes an important regulatory component critical to maintain a proper gut function. This regulatory component regulates the generation of inflammatory processes to fight the dangerous agents, while blocking the responses against the inoffensive agents and preventing excessive tissue damage to maintain the integrity of the epithelial barrier. Indeed, a failure in the regulatory component results in severe consequences for the body's health, such as the inability to resolve infections. In this study, we aim to investigate the interaction between the human body and the enteric bacteria Helicobacter pylori and Clostridium difficile, to bring new insights in the regulatory component of the immune system of the gut. Moreover, the new mechanisms discovered in the regulatory system, might allow the development of new treatments for infectious diseases.

Clostridium difficile is a Gram positive, anaerobic bacterium which produces two potent protein toxins, A and B. The genes for toxins A and B have been previously cloned and sequenced and lie within 1.4 kb of each other. Upstream and downstream boundaries between sequences shared by both toxigenic and nontoxigenic strains and those sequences which are unique to toxigenic strains were established. A toxigenic element was defined in C. difficile strain 10463 which is 19.6 kb in length and is comprised of five open reading frames, including the toxin A and B genes. One of these open reading frames is previously unidentified and is located upstream of toxin B. Products of Polymerase Chain Reaction (PCR) amplification of three regions in the toxigenic element: the upstream boundary, the downstream boundary, and the region between the toxin A and B genes, were all identical in length in six toxigenic strains, indicating that the toxigenic element is conserved among these strains. A short fragment unique to nontoxigenic strains and occupying the same position on the chromosome as the toxigenic element was identified. peR products of this region were identical in length in three nontoxigenic strains. Transcriptional analyses were undertaken using probes to each of the five open reading frames in the toxigenic element. Transcripts were detected for four of the open reading frames which are contiguous and transcribed in the same direction. In addition, a very large transcript, corresponding to the length of the four open reading frames and processing intermediates were detected, indicating that the toxin genes are cotranscribed. A promoter region and processing sites were identified. Sizes were determined for each of the individual transcripts which correspond well with the sizes of the open reading frames. Six toxigenic strains which vary considerably in toxin production were selected for analysis to determine whether DNA sequence variation could account for the observed differences in toxin production. DNA restriction fragment length polymorphisms were examined, toxin-specific transcripts were analyzed, and sequences of regulatory regions were determined and compared. Whereas quantitative differences in toxin-specific transcripts were found among the toxigenic strains, the remaining analyses showed that DNA sequences were conserved among these strains. / Ph. D.

Bakgrunn: Clostridium difficile(CD)er antatt å være den utløsende årsak for 20-30 % av tilfeller med antibiotikaassosiert diaré. CD er den vanligste formen for helsetjenesteervervet diaré, og forår-saker økt sykelighet og dødelighet, samt økte kostnader for helsetjenesten. Desiste tiår er det rappor-tert om endringeri epidemiologien, forårsaket av en ny CD stamme, ribotype 027. Den spres lettere, lager mer alvorlig sykdom og tilbakefall. I Norge har denne stammen til nå ikke vært noe problem. Forekomsten kan reduseres ved en tydelig antibiotikapolitikk, og etterlevelse av anbefalte smitte-verntiltakfor å forebygge CD. Hensikt: Finne ut hvilke smitteverntiltak helsepersonell i sykehuset velger ved håndtering av pasien-ter med CD, og hvor de henter kunnskap om slike smitteverntiltak fra. Metode: En retrospektiv tverrsnittsstudie med spørreskjema til helsepersonell ble gjennomførtvåren og høsten 2011 i Sørlandet sykehus HF (SSHF), sør i Norge. Resultat: 168 helsepersonell deltok i undersøkelsen, fordelt på 59 leger og 109 pleiere. Svarprosen-ten var 94. Antibiotikarestriksjoner sier 94 % av medisinerne og 38 % av kirurgene atde har i sine avdelinger(x²=10.756, p&lt;0.001). Stetoskop brukt i isolat med CD pasienter sier 25 % legerat de spritdesinfiserer og tar med, mens 6 % pleiere sier det samme (x²=22.273, p&lt;0.001). 73 % av leger og 64 % pleiere sier at de både desinfiserer og vasker hendene etter kontakt med CD pasient (x²=6.451, p=0.011). Pasientinformasjon om viktigheten av håndhygiene sier 8 % leger og 36 % pleiere at de alltid gir (p&lt;0.001). Desinfeksjonsmiddel til bruk etter en CD pasient, sier 48 % medi-sinske pleiere og 43 % kirurgiske pleiere at de velgerVirkon (feil). Infeksjonskontroll-programmet (IKP) som kunnskapskilde brukes av 14 % leger og 46 % pleiere, (p&lt;0.001). Kunnskap om smitte-verntiltak ved CD sier 63 % legerog 72 % pleiere at de får fra kolleger. Konklusjon: Helsepersonell har generell kunnskap om smitteverntiltak, men mangler spesifikk kunnskap om smitteverntiltak ved CD. For å møte utfordringen med nye mer spredningspotente CD stammer, må etterlevelsen av smitteverntiltak øke. IKPalene, synes ikke å være tilstrekkelig for å holde helsepersonelloppdatert. Den manglende spesifikke kunnskapen om smitteverntiltak ved CD kan utsette pasienter i sykehuset for smitterisiko / Background: Clostridium difficile(CD) causes 20 %–30 % of all nosocomial infectious diarrhea, resulting in significant morbidity and increasing healthcare costs. Good antibiotic stewardship reduc-es the incidence of CD, andcompliance with infection control measures limits its spread. In recent years, ribotype 027, a new strain of CD, caused several disease outbreaks. Ribotype 027 spreads more easily and increases disease severity and relapse. Thus far, ribotype 027 has caused few prob-lems in Norway. Objective: This thesis aimed to determine whether hospital-based healthcare professionals comply with recommended infection control measures for CD prevention and identify how they learn about such measures. Method: A retrospective cross-sectional survey and questionnaire was performed among healthcare professionals at Sørlandet Hospital, Norway,during the spring and fall of 2011. Results: Survey participants included 168 health professionals (59 physicians and 109 nurses). The response rate was 94 %. Medical doctors (94 %) and surgeons (38 %) said that their clinics impose antibiotic restrictions (x ² = 10.756, p&lt; 0.001). After contact with a CD patient, physicians and nurs-es (73 % and 64 %, respectively) said they disinfect and wash their hands (x ²= 6.451, p&lt; 0.011). Notably, only 8% of physicians and 36 % of nurses always give patients information about the im-portance of hand hygiene (p&lt; 0.001). Even 25 % of physicians and 6 % of nurses reported using ethanol (does not eliminated CD spores) to disinfect stethoscopes before leaving a CD isolation room (x ² = 22.273, p&lt; 0.001). Medical and surgical nurses (48 % and 43 %, respectively) incorrectly used Virkon as a disinfectant in the CD patient’s room. Physicians and nurses (63 % and 72 %, respective-ly) mainly obtain knowledge about infection control measures from colleagues, compared to physi-cians and nurses (14 % and 46 % , respectively) who gain such knowledge from the hospital’s infec-tion control program (p&lt; 0.001). Conclusion: Healthcare professionals have some knowledge about infection control measures, but lack knowledge specific to limiting the spread of CD. Increased compliance with infection control measures is crucial to meeting the challenge of new and more potent strainsof CD. Guidelines alone are likely insufficient to keep healthcare professionals up to date. The lack of specific knowledge about infection control measures for CD may expose hospitalized patients to CD infection / <p>ISBN 978-91-86739-69-0</p>

Objective To describe the use of Lactobacillus by hospitalized patients and to examine its relationship with various Clostridium difficile infection (CDI) related outcomes. Methods The characteristics of Lactobacillus users and non-users and the initiation of Lactobacillus with respect to initiation of antibiotic therapy and CDI treatment were described using national hospital discharge database. The relationships between Lactobacillus use and post-CDI length of stay, mortality, switch of CDI therapy, and readmission were analyzed. Results Lactobacillus users and non-users were different in most characteristics. Metronidazole and fluoroquinolones were the most frequently used antibiotics by Lactobacillus users. They were mainly CDI cases, used multiple antibiotics, extremely ill, and started Lactobacillus five or more days after initiation of antibiotics or CDI treatment. Lactobacillus use was associated with increased length of stay and switching of CDI therapy. Conclusions The true association between Lactobacillus use and CDI remains unclear. This study provides foundation for future research.

Clostridium difficile est responsable de 15 à 25% des cas de diarrhées post-antibiotiques (ATB) et de plus de 95% des cas de colite pseudomembraneuse. Depuis 2003 et suite à l'émergence du clone 027, les ICD sont devenues plus fréquentes et plus sévères. Compte tenu des conséquences, il a été décidé d'explorer en détail et prospectivement cette maladie au Groupement Hospitalier Edouard Herriot L'inclusion des patients a débuté fin février 2011 et devrait s'arrêter fin février 2014. Dans une méta-analyse, nous avons montré que l'ICD communautaire est associée à l'exposition aux mêmes ATB qu'une ICD nosocomiale. Une analyse de la littérature, en utilisant la grille ORION comme outil, nous a permis de synthétiser les connaissances sur la prévention et le contrôle d'ICD en milieu hospitalier. Par la suite sur la base d'une étude rétrospective, le sexe, la CRP et l'exposition aux fluoroquinolones ont été identifiés comme associés à une ICD sévère chez les patients hospitalisés en réanimation. Entre 2011 et 2013, 430 patients ont été inclus dans notre cohorte. L'analyse des données de la prise en charge thérapeutique de 118 cas d'ICD a montré un niveau insuffisant de la connaissance des recommandations actuelles concernant le traitement de cette infection. L'analyse pronostique a montré un taux de mortalité de 19,5% dans les 30 jours qui suivent le diagnostic. L'ICD était indiquée comme une cause principale ou contributive de décès dans quinze cas (65,7% des décédés). Les analyses multivariées ont montré que les facteurs associés au décès sont différents entre les patients avec une ICD et les patients présentant une diarrhée non liée au Clostridium difficile / Clostridium difficile is responsible for almost all cases of pseudomembranous colitis and for 15%-25% of cases of post-antibiotic (ATB) diarrhea. Since 2003 and the emergence of 027 strain, CDI epidemiology is changing, with evidence of rising incidence and severity. In response to the alarming situation we decided to conduct a prospective study at Eduard Herriot Hospital to explore in details this infection. Patient’s inclusion has started in February 2011 and will end in February 2014. In a meta-analysis we found that the risk profiles for antimicrobial classes as risk factors for community-acquired CDI are similar to those described for nosocomial CDI. We used the ORION statement (Outbreak Reports and Intervention Studies Of Nosocomial infection) to synthesize knowledge of interventions to reduce and to control CDI in hospitals. Then in a retrospective study, we found that male gender, rising serum C-reactive protein level, and previous exposure to fluoroquinolones were independently associated with severe CDI in ICU. Between 2011 and 2013, 430 patients were included in our prospective cohort study. Data analysis of 118 cases of CDI showed an inefficient knowledge of current recommendations of CDI treatment. The crude mortality rate within 30 days after CDI diagnosis was 19.5%, with 15 deaths (65.7% of deceased patients) related to CDI. In a multivariate cox regression model, gender, serum albumin, antidiarrheal medications, cephalosporins, peritonitis and septic shock were independently associated with mortality in CDI patients. When diarrhea was not related to C. difficile, mortality was rather associated with cancer and high WBC level

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Thèse d'exercice : Médecine. Médecine générale : Paris 12 : 2005. / Titre provenant de l'écran-titre. Bibliogr. f. 119-131.