As doenças inflamatórias intestinais (DII) são doenças crônicas incapacitantes com significativa morbidade. A Doença de Crohn (DC) e a Retocolite Ulcerativa (RCU) são as maiores representantes. O diagnóstico é baseado na suspeita clínica e complementado por achados endoscópicos, histopatológicos e radiológicos. Como não existe um teste padrão, o diagnóstico destas doenças permanece sendo um problema para gastroenterologistas e patologistas. O desenvolvimento de terapias mais específicas torna cada vez mais importante o diagnóstico preciso para uma escolha terapêutica individualizada. A imunidade inata está ativada de forma semelhante nas duas DII, porém na RCU os linfócitos CD4 TH2 são os principais envolvidos. Estes linfócitos expressam o receptor CD30 na sua membrana plasmática e produzem citocinas que ativam os eosinófilos. Levando em consideração o conhecimento fisiopatológico atual, este estudo tem como principal objetivo avaliar a relevância da expressão do marcador CD30 por imuno-histoquímica, a contagem de eosinófilos e as características histopatológicas no diagnóstico diferencial das DII. Foram avaliados 185 pacientes de um ambulatório especializado em um Hospital Universitário (105 com DC 80 com RCU). O diagnóstico foi estabelecido pelos critérios de Lennard-Jones, revisados por um gastroenterologista especializado e corroborado por cinco anos de seguimento. As biópsias foram realizadas por diversos profissionais na rotina assistencial, porém todas foram analisadas por um patologista com experiência em tubo digestivo. Nenhum paciente estava usando tratamento no momento da coleta das biópsias. As variáveis histopatológicas que demonstraram poder estatístico para auxiliar no diagnóstico diferencial foram a extensão do processo inflamatório para a submucosa, a presença de granuloma, erosões aftóides e variabilidade de acometimento entre os fragmentos. Avaliando estas características em conjunto, foi possível obter uma acurácia de 69.1% para o diagnóstico diferencial entre DC e RCU. Estudando o segmento mais alterado encontrou-se uma mediana de eosinófilos de 42 (25,5 – 63,5), nos pacientes com DC e 107 (67 – 123) nos pacientes com RCU (p< 0.001). Assumindo como ponto de corte um número V a 70 eosinófilos, a sensibilidade foi de 78,3% e a especificidade de 71% favorecendo o diagnóstico de RCU, a área sob a curva ROC foi de 0,767 (IC 95%: 0,696–0,838). A imuno-histoquímica com CD30 demonstrou uma mediana de 3 células CD30+ (2-6) na DC e 33 (24-52) na RCU, demonstrando uma diferença estatística significativa para o diagnóstico diferencial entre as duas doenças (p<0,001). Além disso, nos pacientes com RCU as células CD30+ estavam distribuídas mais frequentemente em agrupamentos nos centros dos folículos linfoides. O ponto de corte determinado pela curva ROC foi de 15 células marcadas (S = 97,5%, E = 94,3%, RV + = 17,1; RV = 0.03, área sob a curva: 0.967, IC 95%: 0.941 - 0.993). Todos estes parâmetros estudados tiveram capacidade discriminatória para o diagnóstico de DC e RCU. Designando um valor para cada variável, baseado no poder estatístico de cada uma e de forma a obter uma soma de 10 pontos no total, foi construído um escore histopatológico para o diagnóstico da DC. Considerando-se a soma das variáveis V 5 obteve-se uma especificidade de 100% e uma sensibilidade de 86,8%. Considerando 4 como ponto de corte do escore, a sensibilidade aumenta para 95,3%, e a especificidade reduz de 100% para 94,9%. Concluindo, o uso rotineiro da avaliação das características histopatológicas descritas em associação com a contagem de eosinófilos e células CD30+ proporciona uma alta acurácia no diagnóstico diferencial entre DC e RCU. Todos os parâmetros estudados são de fácil avaliação tanto por patologistas especialistas quanto generalistas. Sugere-se a realização de um estudo prospectivo para validação deste escore. / Inflammatory bowel diseases (IBD) are chronic disabling diseases with significant morbidity, being Crohn’s disease (DC) and Ulcerative colitis (UC) their greatest representatives. Diagnosis is based on clinical suspicious and complemented by endoscopic, histopathological and radiological findings. However, there is not a gold standard test, so it remained a problem for gastroenterologists and pathologists. The development of more targeted therapies makes it even more important to establish an accurate diagnosis for a better individualized therapeutic approach. Innate immune response is activated in both IBD, but in UC the lymphocytes CD4 TH2-like are mainly engaged, this kind of lymphocytes has CD30 expressed in their plasma membrane and produces cytokines that activate eosinophils. Considering the current pathophysiological knowledge, this study aimed to evaluate the relevance of CD30 expression by immunohistochemical, eosinophil count and histopathological features in differential diagnosis of IBD. A total of 185 patients were evaluated (105 CD/ 80 UC). Patients were followed at a specialized clinic of a university hospital, diagnosed according to Lennard- Jones criteria reviewed by a gastroenterologist expert and corroborated by five years of follow up. Biopsy samples were taken by different professionals in routine care, but analyzed by an experienced gastrointestinal pathologist. None of the patients were using treatment at the time of biopsy. Of all the pathological variables assessed, those that had statistical capacity to assist in the differential diagnosis were extension of the inflammatory process to submucosa, granuloma, aphthous erosion and variability of involvement between fragments. Evaluating these variables together, an accuracy of 69.1% in the differential diagnosis between CD and UC was found. Assessing the most altered sample the median of eosinophils was 42 (25.5 – 63.5) in CD patients and 107 (67 – 123) in UC patients (p< 0.001). Assuming a cutoff V 70 eosinophils, the sensitivity was 78,3% and specificity of 71% favoring the UC diagnosis, the area under the ROC curve was 0.767 (CI 95%: 0,696–0,838). Immunohistochemical CD30+ cells presented with a median of 3 cells (2-6) in CD and 33 cells (24-52) in UC, demonstrating a highly significant statistical difference between the two diseases (p<0.001). Besides, CD30+ cells were distributed most clustered in the center of lymphoid follicles in UC patients. The cutoff determined by ROC curve was 15 (S = 97.5%, E = 94.3%, LR + = 17.1;-RV = 0.03, AUC: 0.967, 95% CI: 0.941 - 0.993). All these parameters studied had discriminatory capacity for diagnosis of CD and UC. A value was assigned to each variable based on the statistical power of each, making a total sum of 10 points to build a histopathological score for the CD diagnosis. Setting the cutoff as V 5 we found a specificity of 100% and a sensitivity of 86.8%. When we consider as cutoff as V 4 points the score sensitivity comes to 95.3%, and reduces the specificity of 100% to 94.9%. In conclusion, the use of routine assessment of the histopathological features described previously in association with the eosinophils and CD30+ cells count provides a high accuracy for CD and UC differential diagnosis. All parameters assessed here are easily performed by pathologists specialists and generalists. The next step seems to be the validation of this score in a prospective study.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-19T12:36:45Z No. of bitstreams: 1 marciavaleriacolli.pdf: 304302 bytes, checksum: de86bd7cd514eef910c8c52ed56c803e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-25T12:18:00Z (GMT) No. of bitstreams: 1 marciavaleriacolli.pdf: 304302 bytes, checksum: de86bd7cd514eef910c8c52ed56c803e (MD5) / Made available in DSpace on 2016-10-25T12:18:00Z (GMT). No. of bitstreams: 1 marciavaleriacolli.pdf: 304302 bytes, checksum: de86bd7cd514eef910c8c52ed56c803e (MD5) Previous issue date: 2007-12-06 / A doença de Crohn (DC) é uma doença inflamatória intestinal idiopática e de expressivo aumento em sua incidência nas últimas décadas. Apresenta manifestações clínicas proteiformes e potencial evolutivo incerto, muitas vezes fatal. Até o momento, não existe qualquer terapia curativa para esta afecção. A azatioprina (AZA) é uma medicação potente e eficaz, que tem sido largamente utilizada no tratamento de pacientes com DC que se tornam dependentes ou refratários aos corticosteróides. Entretanto, esta droga pode apresentar expressiva toxicidade. Para otimizar seus benefícios e minimizar os riscos, é importante que se conheça seu mecanismo de ação, o perfil de efeitos adversos, e como estes podem ser abordados. Objetivos: Avaliar a incidência, abordagem e evolução dos efeitos adversos da AZA no tratamento de pacientes com DC. Material e Métodos: Foram avaliados prospectivamente, de janeiro de 2002 a dezembro de 2006, 106 pacientes (45 homens e 61 mulheres, média de idade de 36,3 anos, variação 12-68 anos), portadores de DC em uso de AZA, atendidos no ambulatório de Doenças Inflamatórias Intestinais do Hospital Universitário da UFJF. Foram registrados por ocasião das visitas ambulatoriais, dados sóciodemográficos relevantes, características clínicas relacionadas à DC, dose e tempo de uso da AZA, e uso de drogas concomitantes direcionadas à DC. Todos os pacientes foram submetidos a rotinas laboratoriais para monitorização da toxicidade durante o tratamento. Assim, foram realizados hemogramas e dosagem de aminotransferases e amilase na inclusão ao estudo. A monitorização subseqüente envolveu hemograma a cada duas semanas durante oito semanas, e depois a cada um ou dois meses durante todo o período da terapia. Dosagem de aminotransferases foi realizada a cada três meses. Além disso, todos os pacientes foram alertados a retornarem prontamente em caso de dor faríngea ou qualquer outro sinal de infecção. Leucopenia foi definida pela contagem de leucócitos abaixo de 4.000 células/mm³ e leucopenia grave quando os mesmos estivessem abaixo de 2.000 células/mm3; trombocitopenia foi considerada quando a contagem de plaquetas foi inferior a 130.000 células/mm3. A AZA foi suspensa ou sua dose reduzida em caso de febre, doenças concomitantes ou toxicidade relacionada à droga. Para fins comparativos, os pacientes foram divididos em grupos com e sem a toxicidade a AZA. Resultados: 56 (52,7%) dos pacientes estudados apresentaram pelo menos um efeito adverso, requerendo redução transitória da dose da droga; 18 (17%) necessitaram suspender definitivamente o uso de AZA, geralmente devido a reações de hipersensibilidade. Náuseas e vômitos, frequentemente leves, ocorreram em 29 (27,4%); a raça negra e aqueles com comorbidades apresentaram mais intolerância gastrointestinal do que os brancos e aqueles sem outras doenças associadas (p=0,04). Leucopenia foi o efeito adverso mais freqüente, ocorrendo em 36 (34%). O tempo de uso de AZA foi maior em pacientes com leucopenia do que nos não leucopênicos (p=0,001), enquanto a dose média de AZA foi menor naqueles com leucopenia comparados aos não leucopênicos (p=0,005). Não houve infecções graves, neoplasias ou óbitos durante o tratamento com AZA. Conclusão: A AZA mostrou ser uma droga relativamente segura no tratamento da DC, desde que seja mantida supervisão clínica e laboratorial periódica durante todo o tratamento. / Crohn’s disease (CD) is an inflammatory bowel disease whose etiology is unknown and that has its incidence increasing significantly during the last decades. Polymorphous clinical manifestations and a doubtful evolution, sometimes fatal, are the more striking features of its natural history. Until nowadays, there is no curative therapy for CD. Azathioprine (AZA) is one of the most efficient and employed drugs in CD patients,especially those who became dependent or non-responders to corticosteroid treatment. However, AZA is a drug which has a potential toxicity and to try to reduce its side effects it is necessary to know its action mechanism as well the list of potential adverse reactions and how to manage them. Aims: To evaluate the incidence, approach and course of AZA side effects in the treatment of CD patients. Methods and Material: From January 2002 to December 2006 106 CD patients were followed prospectively (45 men and 61 women, with a mean age of 36,3 years – range 12-68) in the outpatient Service of Inflammatory Bowel Disease of Universitary Hospital of Federal University of Juiz de Fora, Brazil, and being treated with AZA. Socio-demographic, clinical features of CD patients, AZA doses and time of using, and concomitant other drugs used were registered. All patients were submitted to laboratorial routine for toxicity monitoring during the treatment. Thus, red and white blood cell count and measurement of aminotransferases and amilase levels were analysed at the study beginning; subsequent monitoring included red and white blood cell count at every two weeks for the first two months and lately every four to eight weeks. Aminotransferases levels were determinated every three months. Moreover, all the patients were instructed to search for medical assistance in case of any sign of infections as well as sore throat. Leucopoenia was defined as white blood cells count below 4.000/mm³, and severe as lower 2.000/mm³; trombocytopenia when platelets count were under 130.000/mm³. AZA was stopped or doses reduced when fever, adverse reactions or toxicity AZA-related and associated diseases occurred. In order to comparison, subgroups of patients were studied separately, those with and without AZA-toxicity. Results: 56 (52,7%) patients presented at least one side effect needing transient dose reduction; 18 (17%) had to interrupt definitively the AZA ministration, most of them in consequence of hypersensitivity reactions. Slight nausea and vomits occurred in 29 (27,4%); black race and those with co morbidities have had much more gastrointestinal intolerance than white and those without any other associated disease (p=0,04). Leucopoenia was the most frequent adverse reaction, observed in 36 (34%) and the time of AZA using was higher in patients with leucopoenia than in those nonleucopoenics (p=0,001). However, the mean dose of AZA was lower in patients with leucopoenia than in those non-leucopoenics (p=0,005). No one serious infection, malignant tumors or death occurred during AZA treatment. Conclusions: The AZA use showed to be a reasonable safe drug in CD treatment since that a clinical and laboratorial serious control is performed during all the time of therapy.

The overall aim of this thesis was to study epidemiological and clinical changes in the natural history of Crohn’s disease, its phenotype, the need for surgery and pharmacological therapy over time, as well as the role of faecal calprotectin as a biomarker of pathophysiology and disease course. An increased incidence and prevalence of Crohn’s disease was seen in the period 1963-2010. The proportion of patients with non-stricturing, non-penetrating disease behaviour at diagnosis increased, suggesting that either patients with Crohn’s disease are diagnosed earlier in their disease course today or that the Crohn’s disease phenotype is changing. A decrease in complicated disease behaviour, an increased use of immunomodulators, and a reduced frequency of surgical procedures five years after Crohn’s diagnosis was observed. The decrease in surgery at five years seemed to be explained mainly by a decrease in early surgery within three months from diagnosis, likely reflecting an increased proportion of patients with non-stricturing, non-penetrating disease. This suggests that the introduction of new treatment alternatives alone does not explain the reduction in surgery rates, and an increasing proportion of patients with uncomplicated disease at diagnosis may also play an important role. Subclinical mucosal inflammation, mirrored by increased NFkB activity and increased neutrophil activity (i.e. FC and MPO expression), was observed in healthy twin siblings in both discordant monozygotic and discordant dizygotic twin pairs with IBD. These findings strongly support the hypothesis of an ongoing subclinical mucosal inflammation at the molecular level in healthy first-degree relatives of IBD patients. Baseline FC as well as consecutive FC measurements predict relapse in IBD. The doubling of FC value increased the risk of relapse by 101% in the following three months. This increased risk attenuates with time by 20% for every three month period since the sample was obtained.

Le gène NOD2, impliqué dans les réponses immunitaires innées contre le peptidoglycane bactérien, est étroitement associé à la maladie de Crohn (MC) avec des Odd Ratio(OR) allant de 3 à 36 selon le génotype et a été initialement identifié par des analyses de liaisons génétiques. Les données des familles multiplexes (familles définies par la présence de trois ou plus de trois apparentés au premier degré atteints de MC) issues du registre EPIMAD ont été analysées. Il a été précédemment rapporté que dans les 22 familles multiplexes EPIMAD génotypées pour les 3 mutations majeures du gène NOD2une fréquence élevée de ces mutations du gène NOD2 : R702W, G908R et L1007fs, pouvait expliquer la fréquence élevée de MC dans ces familles. Cependant, quelques familles multiplexes EPIMAD ne présentaient aucune de ces mutations R702W, G908R et L1007fs.Afin d’identifier de nouvelles variations génétiques ayant un effet majeur dans la MC, un protocole de Whole Exome Séquencing (WES) a été effectué sur l’une de ces familles multiplexes EPIMAD (F49M) présentant quatre sujets atteints de MC sur deux générations.Une variation rare du gène NOD2, N1010K, s’est avérée présente chez tous les patients atteints et absente chez tous les sujets contrôles intrafamiliaux . L’évaluation in silico et la modélisation 3D ont mis en évidence un effet délétère hautement probable de la mutation de N1010K suggérant donc fortement qu’elle pourrait être un nouveau facteur de risque majeur impliqué dans la susceptibilité génétique à la maladie de Crohn. Elle pourrait expliquer l’agrégation familiale de la MC dans la famille analysée. La présence d’une maladie plus sévère chez les patients hétérozygotes composites N1010K/L1007fs plaide en faveur de l’effet délétère de la mutation N1010K.En plus de la caractérisation d’une nouvelle mutation rare du gène NOD2, 2 autres variants potentiels ont été identifiés : les mutations D359H et G33V respectivement des gènes BPIFB2 et DEFB132. Les protéines codées par ces gènes sont impliquées dans les mêmes voies de signalisation : la voie de signalisation des défensines ainsi que dans celle du système immunitaire inné. L’évaluation in silico des effet de ces mutations a mis en évidence un effet délétère hautement probable pour D359H et G33V des gènes BPIFB2et DEFB132. Ainsi, on peut supposer que bien que les deux mutations D359H du gèneBPIFB2 et G33V du gène DEFB132, soient localisés sur deux gènes différents impliquées dans les mêmes voies de signalisation, elles pourraient agir ensemble et conduire à un effet dysfonctionnel cumulatif impliqué également dans l’agrégation familiale de la MC dans la famille F49M.Ainsi, Pour la famille F49M, l’agrégation familiale pourrait reposer sur l’accumulation de plusieurs mutations à effet délétère (N1010K, D359H et G33V). / The NOD2 gene, involved in innate immune responses, has been found to be highlyassociated with Crohn’s Disease (CD). EPIMAD multiplex families with three or more CDaffectedmembers were previously reported to be related to a high frequency of NOD2gene mutations : R702W, G908R, and L1007fs. However, some rare EPIMAD CD multiplexfamilies were described without any of the common NOD2 linked-to-disease mutations.In order to identify new genetic variation(s) with amajor effect in CD, whole exomesequencing was performed on available subjects in a multiplex family (F49M), withoutknown common NOD2 mutations and comprising four patients affected with Crohn’s diseaseand three unaffected related subjects on two generations . A rare and, not yet, reportedmissense mutation of the NOD2 gene, N1010K, was detected and co-segregated acrossaffected patients (present in allmembers affectedwith CD and absent in all unaffected familialcontrol subjects). In silico evaluation of the deleterious effect of the mutation and3D modelling highlighted evidences for an adverse effect of the N1010K mutation withregard to the function of the NOD2 protein and the genetic risk of CD.Moreover, N1010Kand L1007fs as a compound heterozygous state in two, more severe CD family membersstrongly suggests that N1010K could be a new risk factor involved in Crohn’s disease geneticsusceptibility.In addition to the characterization of a new rare mutation of the NOD2 gene, 2 otherpotential variants have been identified : the D359H and G33V mutations, respectively, inthe BPIFB2 and DEFB132 genes. The proteins encoded by these genes are involved in thesame pathways : the pathway of defensins and the pathway of the innate immune system.In silico evaluation of the deleterious effect of mutations revealed a potential deleteriouseffect of D359H and G33V mutations. Thus, we could hypothesize that although the two mutations D359H and G33V are located on two different genes but involved in the same signaling pathways, they could act together and determine a cumulative dysfunctional effect also involved as determinants of the familial aggregation of Crohn’s disease in family F49M.Thus, for the F49M family, familial aggregation could be based on the accumulation of several deleterious mutations (N1010K, D359H and G33V).

Bakgrund: Crohns sjukdom är en form av inflammatorisk tarmsjukdom som kännetecknas av kronisk histologisk inflammation, en autoimmun sjukdom som inte är medicinskt botbar men det finns olika medicinska terapier som kan kontrollera symtomen. Det förekommer omväxlande perioder med skov och lugn, operation behövs ibland, medicineringen är konstant och alla dessa faktorer leder till försämrad livskvalitet. Crohns sjukdom skadar ofta ileum men det kan påverka vilket segment av mag-tarmkanalen som helst från mun till perianalt område, lesionerna kan ha formen av fläckar, med vissa delar påverkade medan andra förblir helt normala. Incidensen av Crohns sjukdom i Sverige är mellan 5 och 10 per 100 000 och prevalensen är cirka 200 per 100 000. Medan dödsfallet med Crohns sjukdom minskar, ökar de andra problemen som är förknippade med Crohns sjukdom och detta sätter press på sjukvården. För att hålla sjukdomen under kontroll behöver patienter farmakologisk induktionsbehandling i början av sjukdomen och underhållsbehandling under resten av livet. Just för att behandlingen behövs på lång sikt (livet ut) har det varit absolut nödvändigt att hitta den bästa behandlingsstrategin. Målet med behandlingen är att göra patienten symptomfri med hjälp av medicinering. Mål: Syftet med detta examensarbete var att ta reda på hur tillgängliga biologiska läkemedel ska användas för att få optimal effekt, närmare bestämt om de biologiska läkemedlen ska användas som monoterapi eller i kombination med andra läkemedel vid behandling av Crohns sjukdom. Metod: PubMed-databasen har använts för att söka, välja och analysera fem relevanta randomiserade kontrollerade kliniska prövningar om Crohns sjukdom. Nyckelordet som användes för att hitta alla dessa fem relevanta artiklar var ''Crohn's combination therapy''. Resultat: Resultaten i den första artikeln påpekar att kombinationsbehandling med infliximab + azatioprin hade bättre resultat jämfört med infliximab monoterapi och infliximab monoterapi hade bättre resultat jämfört med azatioprin monoterapi för att uppnå klinisk remission och slemhinneläkning. Den andra artikeln strider mot den första studien och säger att om tillräckliga läkemedelskoncentrationer uppnås och bibehålls genom terapeutisk läkemedelsövervakning med infliximab, kanske kombinationsterapi med tiopuriner inte behövs för att uppnå önskade kliniska resultat. Den tredje artikeln drog slutsatsen att adalimumab var överlägsen tiopuriner i att förhindra endoskopiskt återfall av Crohns sjukdom hos patienter med hög risk för återfall efter operation. Den fjärde artikeln identifierade ingen effektivitetsfördel med kombinationsbehandling med adalimumab + immunmodulatorer jämfört med adalimumab monoterapi. Den femte artikeln drog slutsatsen att vedolizumab i kombination med stabila doser av kortikosteroider inducerade mer effektivt klinisk remission än antingen placebo plus kortikosteroider eller enbart vedolizumab. Slutsatser: En universell slutsats kan inte dras om fördelen med kombinationsbehandling bestående av biologiska och immunsuppressiva läkemedel jämfört med biologisk monoterapi. Olika tillvägagångssätt är nödvändiga för varje enskild patient eftersom medicinen kan vara olika för induktions- och underhållsterapier, sjukdomsvaraktigheten påverkar hur patienten kommer att reagera på mediciner och medicineringen som Crohns sjukdoms-patienter fick tidigare kan också påverka hur patienten kommer att svara på medicinering. / Background: Crohn's disease is a form of inflammatory bowel disease characterized by chronic histological inflammation, an autoimmune disorder that is not medically curable but there are various medical therapies able to control the symptoms. It has periods of exacerbation and calmness, with surgery needed sometimes, the use of medication is constant and all these factors lead to an impaired quality of life. Crohn's disease often damages the ileum but it can affect any segment of the gastrointestinal tract from mouth to perianal area, the lesions may take the form of patches, with some sections affected while others remain perfectly normal. The incidence of Crohn's disease in Sweden is between 5 and 10 per 100,000 and the prevalence is around 200 per 100,000. While the fatal burden is declining, the non-fatal burden is increasing. In order to keep the disease under control, patients require pharmacological induction treatment at the beginning and maintenance treatment for the rest of their life. Because the treatment is needed long-term, finding the best treatment strategy has become imperative. The goal with the therapy is to make the patient symptom-free with the help of medication. Objective: The purpose of this degree project is to find out how biological drugs should be used in order to have optimal effect, more specifically, if the biological medicines should be used as monotherapy or in combination with other medicines in the treatment of Crohn's disease. Method: PubMed database has been used to search, select and analyze five relevant randomized controlled clinical trials about Crohn’s disease. The keyword used to find all these five relevant articles were ''Crohn's combination therapy''. Results: The results in the first article point out that Infliximab + Azathioprine combination therapy had better results compared to infliximab monotherapy and infliximab monotherapy had better results compared with Azathioprine monotherapy in achieving clinical remission and mucosal healing. The second article contradicts the first study and states that if adequate drug concentrations are achieved and maintained through therapeutic drug monitoring with infliximab, combination therapy with thiopurines may not be required to achieve desired clinical results. The third article concluded that adalimumab was superior to thiopurines in preventing endoscopic recurrence of Crohn's disease in patients with high risk of recurrence after surgery. The fourth article identified no efficacy benefit of adalimumab + immunomodulators combination therapy compared to adalimumab monotherapy. The fifth article concluded that vedolizumab in combination with stable doses of corticosteroids induced more efficient clinical remission than either placebo plus corticosteroids or vedolizumab alone. Conclusions: A universal conclusion cannot be drawn regarding the superiority of combination treatment consisting of biological and immunosuppressive medicines compared to biological monotherapy. Different approaches are necessary for every individual patient because the medication can be different for induction and maintenance therapies, the duration of the disease affects how the patient will respond to medication, and the medication that the Crohn’s disease patient took in the past may also affect how the patient will respond to medication.

Crohn’s disease is an inflammatory bowel disease that may involve any part of the gastrointestinal tract with a variety of extraintestinal features. A flare of Crohn’s disease may present as partial small bowel obstruction or peritonitis. Dehydration, infectious agents, and cigarette smoking are some of the factors linked to a relapse of Crohn’s disease. Plesimonas Shigelloides, a bacterium that belongs to the enterobacteriaceae group may rarely lead to a flare of Crohn’s disease. We describe the case of a 31-year-old male with Crohn’s disease who developed a flare triggered by Plesimonas Shigelloides infection presenting as partial small bowel obstruction with ileal narrowing, and regional lymphadenopathy that responded to immunosuppressants.

Introduction. The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are common in Western Europe (200-400 cases /100,000) and associated with substantial morbidity, although mortality is now low. There is presently a great unmet need for novel therapeutics in IBD as present agents are limited by lack of efficacy, toxicity and poor patient acceptance. Recent findings from genome-wide association studies (GWAS) have characterised the genetic architecture of CD and UC. Defects in innate and adaptive immunity have been clearly established, and substantial novel insights into disease pathogenesis have been gained. Over 30 genes / loci are now associated with CD; a number of these, along with a few specific loci, are also associated with UC. The hedgehog (HH) signalling pathway is critical to gastrointestinal development and plays key roles in intestinal and immune homeostasis. Furthermore, in addition to well described roles in tumorigenesis, it is evident that recapitulation of embryonic HH signals play critical roles in response to acute and chronic inflammatory challenge in diverse tissues. Aims. The main aims of the work presented in this thesis were to characterise the expression of key HH signalling components in the healthy and inflamed human intestine, establish whether germline variation in HH genes is associated with IBD and describe the in vitro responses of intestinal epithelial cells to pathogen associated molecular patterns. The WNT pathway, antagonised by HH in the intestine, and two HH target genes (NKX2.3 and CCL20) were also analysed for evidence of association with IBD. Methods. Expression of HH and WNT signalling components was described by immunohistochemistry and microarray analysis in healthy controls (HC), CD, UC, and non- IBD inflamed terminal ileal and colonic samples. Gene-wide haplotype-tagging studies were performed for GLI1 in Scottish, English and Swedish CD and UC, and Scottish early-onset colo-rectal cancer, IHH in Scottish IBD, NKX2.3 in Scottish and UK IBD, and CCL20 in Scottish, Swedish and Japanese IBD. Evidence for association of all HH (n=13) and WNT (n=27) signalling genes in CD was established by analysis of UK GWAS data and metaanalysis from UK, French/Belgium and N American studies. The effect of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on HH signalling was assessed in colonic epithelial cells (SW480). The effect of HH pathway agonists and antagonists on NFκB activity and cytokine expression was analysed in SW480 cells and peripheral blood mononuclear cells (HC and IBD patients) in vitro. Results. The expression of HH pathway ligand is present in the intestinal epithelium and the pathway response network in the lamina propria demonstrating the paracrine nature of HH signalling in the intestine. Immunohistochemical studies and microarray analysis demonstrates that HH pathway activity is decreased in all forms of colonic inflammation studied in man. Variation in Glioma-associated oncogene homolog 1 (GLI1), a key HH transcription factor located at 12q13 (IBD2), was associated with IBD (p<0.0001), UC (p<0.0001) and to a lesser extent CD (p=0.03) in Scotland, a finding replicated in English IBD and UC. This association was attributed to a non-synonymous SNP (rs2228226C→G) with pools odds ratio of 1.194 in meta-analysis of over 5000 individuals from Scotland, England and Sweden (p=0.0002). There was association of this SNP with early-onset colorectal cancer, but of borderline significance (p=0.05). The variant protein (Q1100E) is 50% less active than wild-type protein in vitro. IHH was not associated with CD or UC. Preliminary evidence was produced for association at SUFU (10q24; p=0.005), a GLI1- binding protein, and at the WNT3 / WNT9B locus (17q21; p=0.0005). MDP stimulation of colonic epithelial cells decreased HH pathway activity. Exogenous HH increased expression of CCL20. CCL20 promoter polymorphisms were associated with UC in Japanese patients (p=0.018) but not in Scotland or Sweden. NKX2.3 was associated with IBD in Scotland (UC>CD), but there was insufficient power for fine-mapping of causative variants. Conclusions. Multiple lines of evidence presented here demonstrate that the HH signalling pathway is involved in IBD pathogenesis. In key complementary in vivo studies (conceived by CWL; conducted in collaboration with the Gumucio lab in Ann Arbor) we have demonstrated that Gli1+/- mice develop early, severe colitis with high mortality in response to acute inflammatory challenge. Furthermore, lamina propria antigen presenting cells are identified as the key HH target cells. With HH agonists and antagonists in extensive preclinical and early clinical testing, these studies have real potential to translate into novel therapeutics for patients with IBD.

L'objectif de cette thèse était de développer de nouveaux « donneurs de NO » stables en liant du S-nitrosoglutathion (GSNO) à une structure polymérique. Dans une première étape, les polymères ont été liés au glutathion (GSH) : le chitosan-GSH et l'alginate-GSH ont ainsi été préparés par la « méthode des carbodiimides » et dans une deuxième étape, les polymères finaux [SNOC (S-nitrosoglutathione-oligosaccharide-chitosan) et SNA (S-nitrosoglutathione-alginate)] ont été préparés par nitrosation des deux conjugués précédent. La quantité de NO fixée a été déterminée par les méthodes Griess et Saville. L’aptitude des polymères à libérer du NO et à passer la barrière intestinale [SNOC et SNA] a été évaluée dans une chambre d’Ussing. Nous avons obtenu des polymères avec des quantités variables de NO en fonction de la méthode utilisée (159 µmol de NO/g à 525 µmol de NO/g pour le SNOC ; 174 µmol de NO/g à 468 µmol de NO/g pour le SNA). Le SNOC était stable pendant au moins 6h et le SNA pendant au moins 10h. Enfin, nous avons essayé de mettre au point des microparticules de GSH et GSNO par spray drying avec de l’Eudragit ® FS 30D gastro-résistant. La caractérisation des microparticules a été réalisée par microscopie électronique à balayage (SEM), par diffraction X (PXRD) et par spectroscopie infrarouge (FTIR). Les essais de libération in vitro ont été réalisés dans un tampon (pH 1,2, 3, 6, 6,8 et 7,4). Les microparticules étaient chargées négativement avec une taille moyenne allant de 5 à 7 µm. La formulation était stable à pH acide mais a montré une libération rapide à pH basique ; elle pourrait donc servir de système de délivrance du NO au niveau intestinal. / The aim of the thesis was to develop novel and stable NO-donors by linking S-nitrosoglutathione (GSNO) to a polymer backbone. In the first step, chitosan-GSH and alginate-GSH conjugates were prepared by a carbodiimide reaction and in the second step SNOC (S-nitrosoglutathione-oligosaccharide-chitosan) and SNA (S-nitrosoglutathione-alginate) were prepared by the nitrosation of both conjugates respectively. The amount of NO was determined by Griess and Saville methods. Stability and ex vivo experiments of SNOC and SNA were performed in an Ussing chamber through rat intestine. We obtained polymers with different amount of NO (i.e. 159 µmol of NO/g to 525 µmol of NO/g for SNOC; 174 µmol of NO/g to 468 µmol of NO/g for SNA) depending upon the procedure of nitrosation. SNOC was stable for at least 6h and SNA for at least 10h. Also, we aimed to develop spray dried microparticles of GSH and GSNO based on Eudragit® FS 30D polymer. The microparticles were characterized by scanning electron microscopy (SEM), X-ray diffraction (PXRD), infrared spectroscopy (FTIR) and in vitro release studies were performed in different pH conditions (pH 1.2, 3, 6, 6.8 and 7.4). The microparticles were negatively charged with mean particle size ranging from 5 to 7 µm. The formulation was stable and was resistant to acidic pH but showed rapid release in basic pH; hence, they can be used as colon specific drug delivery systems for the treatment of Crohn’s disease. We think that these formulations could be used in animal models in the treatment of Crohn’s disease.

Depuis les cinquante dernières années, l'incidence de la maladie de Crohn,maladie chronique inflammatoire de l'intestin (MICI) a plus que doublée. C'est une pathologie multifactorielle et idiopathique utilisant des traitements comme des anti-inflammatoires, des corticoïdes, des immunosuppresseurs et plus récemment les anti-TNF.Ils présentent des limites avec une intolérance aux principes actifs et une résistance au traitement conduisant à un échec thérapeutique. L’objectif de ce travail sera de développer une forme orale tout en minimisant le risque d’effets secondaires en améliorant la biodisponibilité. Des techniques d'encapsulation protégeant les principes actifs extraits de plantes comme le Resvératrol, le Bêta-carotène, et la Curcumine seront réalisées. Quatre types de synthèse seront respectivement utilisés : la micro-encapsulation , la nanoémulsion, et la synthèse de nanoparticules solides lipidiques (SLN), ainsi que la squalénisation.Une comparaison des effets anti-inflammatoires des traitements conventionnels versus les vecteurs synthétisés au cours de cette étude pourra permettre de conclure sur la contribution originale de ce projet. / For the last fifty years, the incidence of Crohn's disease, a chronic inflammatory bowel disease (IBD) has more than doubled. It is a multifactorial and idiopathic pathology using treatments such as anti-inflammatories, corticosteroids, immunosuppressants and more recently anti-TNFα.They have limits with intolerance to active principles and resistance to treatment leading to therapeutic failure . The goal of this work will be to develop an oral form while minimizing the risk of side effects by improving bioavailability. Encapsulation techniques protecting the active ingredients extracted from plants such as Resveratrol, Beta-carotene, and Curcumin will be performed. Four types of synthesis will be used respectively: micro-encapsulation, nanoemulsion, and the synthesis of lipid solid nanoparticles (SLN), as well as squalenization. A comparison of the anti-inflammatory effects of conventional treatments versus the vectors synthesized during this study. The study may conclude on the original contribution of this project.

The intestinal mucosa is made up of complex secreted mucus layer consist of mainly mucin 2 (MUC2) and antimicrobial components that defend the underlining cellular barrier from intrusion by luminal microbiota and toxins. In inflammatory bowel diseases (IBD), the mucosal integrity is compromised. This can result from a combination of altered host genetics, gut immune responses and environment factors. However, it is the presence of intestinal bacteria that is central to the pathogenesis of IBD. As part of the dynamic gut microbial flora, mucolytic bacteria produce a wide range of glycosidases that are able to remove the outer oligosaccharide chains of MUC2, which allow other luminal bacteria to further degrade the mucin. We hypothesised that increased mucolytic bacteria will cause excessive degradation of the mucus layer, which in turn, allow more luminal bacteria to be in close proximity to the underlining epithelial cells resulting in inflammation. Consistent with our group’s previous semi-quantitative bacterial 16S rRNA gene clone library analysis, we found increased Ruminococcus gnavus in non-inflamed ulcerative colitis (UC) mucosa. R. gnavus was previously isolated by others based on its mucolytic property. In this study, we quantify total mucosa-associated bacteria and mucolytic bacteria, namely, R. gnavus, R. torques, Akkermansia muciniphila and bifidobacteria. We were able to show quantitatively that total mucosa-associated bacteria were increased in IBD. There was also a population shift in the mucosa-associated mucolytic bacteria, which were increased overall. There was significantly more R. gnavus in non-inflamed IBD biopsies. For the first time, we were also able to demonstrate that R. gnavus can degrade human MUC2 in vitro. To examine whether the numerical association of R. gnavus in IBD does have functional influence on intestinal inflammation and Paneth cell antimicrobial peptide gene expression, we fed mice with R. gnavus. Interestingly, R. gnavus feeding did not result in histological or molecular evidence of gut inflammation; however, it was able to specifically induce Paneth cell cryptdins and lysozyme P genes expression in 3 week old, antibiotic pre-treated C57BL/6 mice. This demonstrated that R. gnavus is not a pathogenic bacterium, which will directly cause colitis. However, the increased Paneth cell response suggested the need for host innate defence when R. gnavus is increased. Other than bacterial degradation, altered host genetics will also influence the mucus barrier. There is evidence to suggest that the MUC2 gene is highly unstable and is susceptible to gene copy number variation (CNV). Therefore, we hypothesised that MUC2 CNV is present, which may result in altered oligomerisation of the MUC2 glycoprotein causing endoplasmic reticulum stress of the goblet cells that appears to be characteristic of UC. Currently, our data partly support the presence of MUC2 CNV. However, further investigation is required to verify the MUC2 CNV identity. Only then can a high throughput methodology be designed to screen a large population for any association with IBD.