IL-23 is an essential mediator of chronic intestinal inflammation in experimental models of colitis. Polymorphisms in the IL23R locus are associated with IBD susceptibility in humans. The biological activity of IL-23 has been linked to Th17 cells but little is known about the cellular and molecular mechanism by which IL-23 drives intestinal inflammation. The work presented herein has identified that direct IL-23 signalling into CD4+ T cells was not only required for the accumulation of Th17 cells in the intestine but also modulated their phenotype. Through direct cell intrinsic effects on T cells, IL-23 drove the emergence of an IL-17A+IFN-γ+ population of T cells that co-expressed RORγ and T-bet. Interestingly, we found that expression of RORγ but not T-bet by T cells was required for the development of intestinal inflammation. Furthermore, colitis induced by T-bet deficient T cells was dependent on IL-17A, and showed a unique inflammatory phenotype, thus demonstrating that pathogenic intestinal Th17 responses can develop independently of T-bet. In addition, using transcriptional profiling we identified a core set of genes that is regulated by direct cell-intrinsic IL-23 signals into intestinal CD4+ T cells. This revealed a previously unrecognised role for IL-23 in suppressing Th2 associated genes, such as GATA3 and IL-33R. Functional experiments demonstrated that expression of GATA3 in CD4+ T cells limited their colitogenic potential, suggesting that IL-23-mediated inhibition of GATA3 might contribute to the development of intestinal inflammation. Finally, we described a novel function for IL-33 as a factor that promotes Foxp3+ iTreg differentiation in vitro and in vivo through direct effects on T cells. This activity of IL-33 was inhibited in the presence of IL-23, providing a mechanistic link for the known role of IL-23 in restraining iTreg generation. Collectively, these data suggest that IL-23 promotes acquisition of a pathogenic effector T cell phenotype through multiple mechanisms. This indicates that therapeutic blockade of IL-23 is likely to reduce pro-inflammatory mediators while also facilitating the expansion of regulatory pathways that might help to re-establish intestinal homeostasis.

Leukocyte extravasation is mediated in part by tissue specific chemotactic cytokines (chemokines) and specific chemokine receptors expressed on the surface of circulating cells. C-C chemokine ligand CCL25 is expressed exclusively in the intestine and thymus and mediates chemotaxis by cells expressing receptor CCR9. This chemokine and receptor pair may be relevant in the pathogenesis of intestinal inflammation, in diseases such as Crohn’s disease (CD) and coeliac disease. In this thesis I investigated CCR9 expression in situ, in tissues affected by intestinal inflammation, and also examined the effects of CCR9 antagonist treatment in patients. In vitro I investigated CCR9 function using human peripheral blood T cells enriched for CCR9 by cell sorting or all-trans retinoic acid treatment. Using tissues collected as part of a clinical trial in CD testing CCR9 antagonist, CCX282-B, I investigated ways of measuring if treatment reduced the number of CCR9 expressing cells in the intestinal mucosa. However, in situ staining for CCR9 by immunohistochemistry was unsuccessful, and in this thesis, I explored reasons why this might be the case. Treatment with CCX282-B did however, show a tendency to reduce T cell density in the intestinal mucosa, although results were highly variable between individuals. In an examination of human CCR9 function in vitro I demonstrate for the first time that CCL25 stimulates CCR9 surface internalization. These data clarify the observation that CCR9 staining by IHC produces poor results in tissues where ligand is abundant, such as the intestine and thymus. I describe a novel technique for measuring calcium flux in two populations simultaneously by flow cytometry, which confirmed that in a heterogeneous population of cells, only CCR9 expressing cells respond to CCL25 by calcium flux. Variability in clinical trials is partly created by the use of concomitant medications, and in CD, corticosteroids are widely used. For the first time I show that glucocorticoids (GC) impair CCR9 mediated chemotaxis, calcium flux and intracellular signalling without changes to CCR9 mRNA and surface protein expression. Reduced CCR9 mediated signalling was accompanied by an enhanced expression and function of co-expressed CXCR4, demonstrating that the effects of GC were receptor-specific and not mediated by non-specific toxicity or inhibition of cell signalling. In a second study CCX282-B was tested in patients with coeliac disease, and in this trial, there was no reported concomitant use of GCs. It was confirmed that dietary gluten stimulates significant T cell recruitment to the intestinal mucosa with a pronounced accumulation of intraepithelial lymphocytes (IEL) and a rise in the frequency of FoxP3 expressing cells. Patients on CCX282-B had lower IEL counts, and an equivalent proportion of FoxP3 expressing T cells, suggesting that CCR9 blockade restricted the recruitment of effector T cell subsets. This thesis confirms that the accumulation of T cells is central to inflammation in the intestine and that modulating chemokine receptor function may affect this. Furthermore, this thesis demonstrates that the function of CCR9 is suppressed by GCs, which are widely used therapeutically and therefore could identify a novel mechanistic basis for their activity in CD.

Bakgrund: Majoriteten av de som drabbas av inflammatorisk tarmsjukdom är kvinnor. Att drabbas vid ung ålder kan öka problematiken. Det finns luckor i forskningen om de psykosociala faktorernas inverkan. Kronisk sjukdom skapar ofta lidande och för att kunna hantera det på ett värdigt sätt krävs kunskap och förståelse. Patienternas erfarenheter om sin sjukdom genererar en bättre inblick och ökad förståelse för sjuksköterskan. Kunskapen hjälper sjuksköterskor att erbjuda god vård för att patienten ska kunna uppleva en så bra livskvalité som möjligt. Syfte: Syftet med denna studie var att belysa unga kvinnors erfarenheter av att leva med en kronisk inflammatorisk tarmsjukdom. Metod: Datamaterialet analyserade med kvalitativ innehållsanalys. Data samlades genom 10 bloggar. Resultat: Ur analysen framträdde fyra kategorier: Vardagen begränsas, Lära sig leva med sjukdom, Kosten styr, Nöjda trots en bristande sjukhusorganisation och åtta underkategorier. Konklusion: Sjuksköterskans ökade kunskap och förståelse kring unga kvinnors erfarenheter av att leva med inflammatorisk tarmsjukdom är av stor vikt för att kunna erbjuda en god och individanpassad omvårdnad. Sjuksköterskans förståelse för patienten bidrar även till en stärkt vårdrelation och sjuksköterskan kan då ge den stöttning patienten efterfrågar.

The mammalian intestine harbors an estimated 100 trillion microorganisms, which normally maintain a mutually beneficial relationship with the host. The intestinal epithelium consists of a single layer of intestinal epithelial cells (IECs) that provides a physical barrier as well as innate immune defense, preventing this vast community of microbes from entering host tissues. Secretory immunoglobulin A (SIgA) acts as the first line of antigen-specific immunity at the interface between the gut microbiota and the intestinal epithelium. Polymeric IgA secreted by plasma cells in the intestinal lamina propria is transported across IECs by the polymeric immunoglobulin receptor (pIgR). Defects in epithelial barrier and immune functions can lead to infections with opportunistic and pathogenic microbes and contribute to the etiology of inflammatory bowel disease (IBD). Here we investigate the ability of IEC biomarkers to define the mechanism and severity of intestinal inflammation, as well as provide insight into the function of IEC in regulating intestinal homeostasis and inflammation. Importantly, down-regulation of pIgR expression was a common feature in human IBD and mouse models of experimental colitis. One molecule of pIgR is consumed for every molecule of SIgA transported, thus high expression of pIgR is required to maintain sufficient supply of SIgA. Accordingly, we investigate the mechanisms by which IECs regulate pIgR expression in response to colonic bacteria. Cross-talk between the microbiota and IECs is mediated by pattern recognition receptors, including Toll-like receptors (TLR), leading to expression of gene products that enhance epithelial barrier function and innate immunity. The cytoplasmic adaptor protein MyD88 transduces signals from TLRs that recognize bacterial products. We show that pIgR induction by colonic bacteria is dependent on TLR4-MyD88 activation of NF-κB signaling. We examined the role of epithelial-specific MyD88 signaling in antibacterial immunity and epithelial expression of key gene products that participate in innate immunity in the gut by generating mice with an IEC-targeted deletion of the Myd88 gene (MyD88ΔIEC). MyD88ΔIEC mice display immunological and antimicrobial defects resulting in increased susceptibility to experimental colitis. We conclude that cross-talk between bacteria and IECs via MyD88-dependent signaling is crucial for maintenance of gut homeostasis.

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.

<p>UVOD: Blastocistis hominis (Bh) je najrasprostranjeniji protist na na&scaron;oj planeti, ali pri tome najkontraverzniji. Infekcija Bh počinje ingestijom hrane ili tečnosti koja je kontaminirana cističnom formom Bh. Nakon gutanja, iz ciste se razvijaju u debelom crevu čoveka vakuolarne forme protista. Fekalno - oralni prenos je najče&scaron;ći put &scaron;irenja infekcije. Oboljenje koje Bh izaziva kod ljudi naziva se blastocistoza. Najče&scaron;će inficirani imaju gastrointestinalne tegobe, pre svega bol u trbuhu i proliv. Blastocistoza se danas povezuje sa dva klinička entiteta koji predstavljaju poremećaj rada creva, odnosno sindromom iritabilnog creva i hroničnom inflamatornom bolesti creva (HIBC). CILJ RADA I HIPOTEZE: Predmet istraživanja je da se utvrdi povezanost prisustva infekcije Blastocistis hominisom i postojanja zapaljenja sluzokože debelog creva (kolitisa) kod dece sa gastrointestinalnim tegobama, zatim da se utvrdi udeo dece sa posebnom formom kolitisa, hroničnom inflamatornom bolesti creva, među inficiranim Blastocistis hominisom, a da bi se omogućilo bolje razumevanje blastocistoze kod dece. Osnovne hipoteze u istraživanju su statistički značajno veća učestalost pojave kolitisa i hronične inflamatorne bolesti creva kod dece uzrasta od 1 meseca do 18 godina, hospitalizovane zbog bola u trbuhu i/ili proliva koji su inficirani Blastocistis hominisom, kao i statistički značajno veća učestalost kolitisa u odnosu na hroničnu inflamatornu bolest creva u istom uzorku. MATERIJAL I METODE: Prospektivnim ispitivanjem su obuhvaćeni pedijatrijski bolesnici, hospitalizovani na Odeljenju za gastroenterologiju, hepatologiju i ishranu, Instituta za zdravstvenu za&scaron;titu dece i omladine Vojvodine, zbog bola u trbuhu i/ili proliva, iz čije stolice je dokazan Blastocistis hominis. U toku ispitivanja primenjene su standardne metode uzimanja anamneza od bolesnika, fizički pregledi, odgovarajuće standardne laboratorijske analize krvi i stolice, ultrazvučni pregled abdomena, kolonoskopija i patohistolo&scaron;ki pregled biopsija debelog creva. Svi bolesnici su lečeni metronidazolom u trajanju 10 dana, prema važećim terapijskim protokolima. REZULTATI: Ispitivanjem je obuhvaćeno 102 bolesnika, koji su an osnovu patohistolo&scaron;kog nalaza podeljeni u tri grupe: 1. Grupa (bolesnici koji nemaju kolitis, obuhvatila je 4 bolesnika (4.4%)), 2. Grupa &ndash; (bolesnici koji imaju nespecifični kolitis, obuhvatila je 56 bolesnika (56.55%)) i 3. Grupa &ndash;(bolesnici koji imaju hroničnu inflamatornu bolest, obuhvatila je 42 bolesnika (42.41%)). Među ispitanicima je bio podjednak broj dece mu&scaron;kog i ženskog pola, odnosno 51 dečak i 51 devojčica. Uzrast ispitanika koji imaju infekciju Blastocistisom hominisom se kretao u interval od 11 meseci do 17 godina i 7 meseci. Medijana je iznosila 12.54 godine, a prosečna starost 11.25 godine. Blastocistoza nema sezonski karakter (&chi;2=0.667; df=3; p=0,881). Značajno vi&scaron;e inficiranih Blastocistis hominisom živelo u kući, nego u stanu i posedovalo domaće životinje i/ili kućne ljubimce, ali ne postojanje odgovarajućih higijenskih uslova, kanalizacije i vodovoda nije prediktivni faktor za razvoj infekcije Blastocistis hominisom, kao ni pohađanje kolektiva ili život u ruralnom sredinama. Stariji uzrast deteta (p=0,020) i život u kući (p = 0,033) su prediktivni faktori za pojavu hronične inflamatorne bolesti creva kod dece sa kolitisom. Deca sa blastocistozom su imala antropometrijske parametre u granicama normale.Ispitanici najče&scaron;će bili primljeni u bolnicu pod djagnozom gastroenterokolitisa, zbog proliva i bola u trbuhu, a da prisustvo gastrointestinalnih tegoba i prisustvo op&scaron;tih znakova infekcije nisu jedan od sigurnih kliničkih značajnih znakova infekcije Blastocistis hominisom. Prisustvo patolo&scaron;kih primesa u stolici nije jedan od sigurnih klinički značajnih znakova infekcije Blastocistis hominisom.Na osnovu laboratorijskog, kliničkog i endoskopskog skora za aktivnost HIBC većina bolesnika je imala umerenu aktivnost.Inficirani sa Bh imaju najče&scaron;će C-reaktivni protein u okvirima refentnih vrednosti, izuzev ukoliko nemaju i HIBC. Povi&scaron;ena sedimentacija eritrocita je karakteristična za bolesnike sa HIBC. Oboleli od blastocistoze imaju najčesce imunoglobulin A, leukocite, neutrofile i eozinofile u krvi u referentnim granicama.Vrednosti feremije upućuju da je većina ispitanika bila anemična, a naročito deca koja su imala i infekciju sa Bh i HIBC. Kod bolesnika sa blastocistozom, postojanje pozitivnog testa na okultnu krv u stolici, treba da pobudi sumnju na udruženu HIBC. Ispitanici sa infekcijom Bh i sa HIBC su imali najče&scaron;će kvantitativno veći broj Bh u stolici. Mezenterajalni limfadenitis i splenomegalija su nespecifični ultrazvučni nalaz kod inficiranih sa Bh, iako su bili najče&scaron;će opisane patolo&scaron;ke promene na ultrazvuku abdomena. Zaključujemo da su ispitanici najče&scaron;će imali nespecifične endoskopske promene i patohistolo&scaron;ke promene u debelom crevu. Metronidazol je bezbedan i efikasan, u dozi 15-50 mg/kg/dan, u trajanju od 10 dana, u terapiji infekcije sa Bh kod dece. ZAKLJUČAK: Deca inficirana sa Bh imaju najče&scaron;će colitis od patolo&scaron;kih promena na debelom crevu, bez značajne razlike između nespecifičnog kolitisa i HIBC. Značajno manje inficiranih sa Bh ima uredan kolonoskopski nalaz.Utvrđivanja značaja Blastocistis hominisa u nastanku kolitisa i hronične inflamatorne bolesti creva kod dece, doprinosi prihvatanju Blastocistisa hominisa kao patogena i ukazuje na nephodnost njegovog lečenja.</p> / <p>INTRODUCTION: Blastocystis hominis (Bh) is the most outspread protist on our planet, but also the most controversial. Infection Bh starts by digestion of the eaten food or liquid which has been contained by a cyst form Bh. After swallowing, from the cyst they grow (progress) in the colon of the human, with a vacuolar form of a protest. Oral transmission is the most common way of spreading the infection. The disease caused by Bh on humans is called blastocystisis. In most cases the infected humans have gastrointestinal complaints, the most common are abdominal pain and diarrhea. Blastocystis is nowadays connected to two clinical disease, the irritable bowel syndrome and inflammatory bowel disease (IBD). THE AIM AND HYPOTHESESS: The subject of research is to establish the connection between the presence of the infection Bh and the existence of mucosal inflammation of the colon in children with gastrointestinal complaints, as well as to establish the group of the children with a special form of colitis, inflammatory bowel disease and the ones infected by Bh, wich would insure better understanding of the blastocystosis in children. The basic hypothesis in the study were statistically significantly higher incidence of chronic colitis and inflammatory bowel disease in children aged 1 month to 18 years, hospitalized for abdominal pain and/or diarrhea who are infected Bh, as well as significantly higher incidence of colitis compared in chronic inflammatory bowel disease in the same sample. MATERIALS AND METHODS: The prospective study included pediatric patients with abdominal pain and/or diarrhea, and stool positive on Bh, that have been hospitalized on the Department for gastroenterology, hepatology and nutrition, in the Institution for Health Care of Children and Youth in Vojvodina. The standard testing methods were used: anamnesis, physical examination, laboratory analysis of blood and stool, ultrasound examination of the abdomen, colonoscopy and histopathological examination of the biopsy of the colon. All patients have been treated with metronidazole for 10 days, according to the applicable protocols. RESULTS: The study included 102 patients, which are divided into three groups : 1. group (patients that have no colitis, included 4 patients (4.4%)), 2. group (patients with unspecified colitis, included 56 patients (56.55%)) and 3. group (patients with inflammatory bowel disease, included 42 patients (42.41%)). Among them, there was an equal number of children that were male and female, 51 boys and 51 girls. Age of respondents who have Bh infection ranged from 11 months to 17 years and 7 months. The median is 12.54 years, and the average age of 11.25 years. Blastocistosis no have seasonal character (&chi;2 = 0.667, df = 3, p = 0.881). Significantly more infected Blastocistis hominid lived in the house, but in an apartment owned and domestic animals and / or pets,yet the existence of appropriate hygiene, sanitation and water supply is not a predictive factor for the development of infection Bh, as well as attending the collective or life in rural areas . The older child&#39;s age (p = 0.020) and life at home (p = 0.033) were predictive factors for development of inflammatory bowel disease in children with colitis. Children with blastocistosis had anthropometric parameters within normal limits. Respondents most frequently been admitted to hospital under diagnosis gastroenteritis due to diarrhea and abdominal pain, and that the presence of gastrointestinal symptoms and general signs of infection are not a significant clinical signs of infection Bh. The presence of pathological findings in stool is not one of reliable signs of clinically infection Bh. Based on laboratory findings, clinical and endoscopic activity score for IBD most patients had moderate activity of desease. Children with Bh infection usually have normal C-reactive protein in terms of value, unless if have IBD. Elevated erythrocyte sedimentation rate is characteristic of patients with IBD. Children with blastocistosis usually have normal level of Immunoglobulin A, leukocytes, neutrophils and eosinophils. Serum iron indicate that most subject were anemic, especially children who have had an infection with the Bh and IBD.Children with blastocistosis, the existence of a positive test for occult blood in the stool, should arouse suspicion of association IBD. Subject with IBD had mostly quantitatively greater number of Bh in the stool. Mesenterial lymphadenitis and splenomegaly are non-specific ultrasound findings in infected with Bh, although they were usually described pathological changes in abdominal ultrasound. This is to conclude that the subject usually had colitis and IBD changes in endoscopic and histopathological changes in the colon. Metronidazole has beem proved safe and effective, at 15-50 mg/ kg/day for 10 days in the treatment of infections in children with Bh. CONCLUSION: Children infected with Bh colitis usually have pathological changes in the large intestine, with no significant difference between the non-specific colitis and inflammatory bowel disease. Significantly less infected with Bh has a normal colonoscopy findings. Confirmed the importance of Bh in the development of chronic colitis and inflammatory bowel disease in children, increase public acceptance Blastocistisa hominis as pathogens and points to the necessity of treatment.</p>

Elevated levels of hyaluronan are associated with numerous inflammatory diseases including ulcerative colitis, Crohn's disease and wound healing. Various proinflammatory cytokines have shown to influence hyaluronan expression in cells originating from connective tissue. The overall purposes of this study included: 1. To determine the effects of IL-1β stimulation on HA and HAS2 steady state transcript levels and the signaling pathways involved in its effects. The signaling pathways utilized by proinflammatory mediators to modulate hyaluronan expression have only begun to be elucidated. In this aim, the effects of IL-1β on hyaluronan and HAS expressions in jejunum-derived mesenchymal cells were determined. Inhibition studies were utilized to determine the signaling pathways involved. The overall hypothesis of this aim was that stimulation of jejunum-derived mesenchymal cells with IL-1β activates the mitogen activated protein kinase pathways resulting in increased HAS2 steady state transcript and hyaluronan levels.Results: The results suggest that IL-1β induction of HAS2 expression involves, in part, the mitogen activated protein kinase signaling pathways that act in concert thus leading to an increase in expression of hyaluronan by jejunum-derived mesenchymal cells.2. To determine the effects of dexamethasone on IL-1β mediated increase in hyaluronan and HAS2 expressions and the mechanisms utilized by this glucocorticoid. Glucocorticoids are a mainstay treatment for the inflammatory component of inflammatory bowel disease. Given the recent evidence demonstrating increased hyaluronan in inflamed tissue from patients affected with inflammatory bowel disease, the objective of this aim was to determine the effect of dexamethasone on IL-1β-mediated induction of hyaluronan. The hypothesis of this aim was that pre-treatment with dexamethasone suppressed the ability of IL-1β to increase HAS2 transcript and hyaluronan levels via inhibition of the p38 MAP kinase pathway. Results: Pre-treatment with dexamethasone inhibited IL-1β-mediated hyaluronan and HAS2 induction by blocking the activation of the p38 MAP kinase pathways. 3. To identify the transcriptional and post-transcriptional mechanisms utilized by IL-1β to upregulate HAS2 steady state transcript levels. Very little is known about transcriptional and post-transcriptional regulation of the hyaluronan synthase 2 gene. In this aim, 5' and 3' mapping, luciferase analyses and actinomycin D studies were used to determine the transcriptional and post-transcriptional mechanisms utilized by IL-1β to regulate HAS2 steady state transcript levels. The hypothesis of this aim was that IL-1β used post-transcriptional mechanisms to regulate the HAS2 gene.Results: Dermal fibroblasts were used to find the 5'- and 3'-termini of the HAS2 message. Promoter constructs extending approximately 1 kb upstream from the transcription start site demonstrated no IL-1β response. Blocking protein synthesis prior to the addition of IL-1β dramatically increased HAS2 steady state transcript levels, while inhibition of transcription suppressed the effect of IL-1β on HAS2. Northern blot analysis revealed that cycloheximide and IL-1β exerted differential effects on the two HAS2 transcripts.

La présente étude a pour objectif de mettre en évidence l‘intérêt du suivi thérapeutique pharmacologique (STP) des métabolites thiopuriques en vue de l‘optimisation du traitement par l‘azathioprine chez les enfants atteints de maladie inflammatoire de l‘intestin et d‘hépatite autoimmune. Les travaux réalisés nous ont permis de montrer, en utilisant une analyse multi-niveaux, une corrélation significative entre la dose d‘azathioprine et les concentrations en 6-TGN et Me6-MPN ainsi qu‘avec le ratio Me6-MPN/6-TGN confortant l‘utilisation des métabolites pour ajuster la posologie d‘azathioprine chez les enfants présentant une maladie inflammatoire de l‘intestin. Différents facteurs pouvant modifier les concentrations de métabolites thiopuriques ont été identifiés. La co-administration d‘infliximab a conduit à une augmentation significative des concentrations de 6-TGN. Des concentrations plus faibles de 6-TGN ont été observées chez les jeunes enfants suggérant l‘influence de l‘âge sur le métabolisme ou sur l‘absorption de l‘azathioprine. Nous avons également montré qu‘une concentration de 6-TGN supérieure à 405 pmol/8.108RBCs chez des patients ayant une activité TPMT normale et pour lesquels la rémission clinique n‘a pas pu être obtenue en l‘absence de stéroïdes, était prédictive d‘une résistance à l‘azathioprine. Un seuil de 250 pmol/8.108RBCs en 6-TGN est significativement associé à une meilleure réponse thérapeutique. D‘autre part, une corrélation a été observée entre les métabolites thiopuriques et la leucopénie. Chez les enfants atteints d‘hépatite autoimmune, une corrélation positive entre la dose d‘azathioprine et les concentrations de métabolites a été retrouvée. L‘importante variabilité inter-individuelle dans les concentrations en métabolites thiopuriques et dans la réponse thérapeutique a été confirmée démontrant l‘intérêt d‘une individualisation de la thérapeutique. L‘instauration d‘un STP des métabolites thiopuriques associé au suivi hématologique, à la détermination de la TPMT et au suivi clinique paraît justifié afin d‘optimiser la thérapeutique par l‘azathioprine dans la population pédiatrique / The present study aimed to investigate the usefulness of thiopurine metabolite monitoring in pediatric inflammatory bowel disease (IBD) and Autoimmune Hepatitis (AIH) for optimizing azathioprine therapy. Using multilevel analysis, we demonstrated for the first time the significant positive correlations between the weight-based azathioprine dosage and the 6-thioguanine nucleotide (6-TGN) and 6-methyl-mercaptopurine (6-MeMPN) levels as well as 6-MeMPN/6-TGN ratio, supporting the use of metabolites to adjust dosing in IBD children. Other factors affecting metabolite levels were also identified. Co-administration of infliximab resulted in a significant increase in 6-TGN levels. Younger children exhibited lower metabolite levels, suggesting the influence of age on metabolism/absorption of azathioprine. We also reported that a 6-TGN level above 405 pmol/8.108RBCs in IBD children with normal TPMT activity who did not achieve steroid-free clinical remission was predictive of azathioprine refractoriness. A cut-off of 250 pmol/8.108RBCs for 6-TGN was found to be significantly associated with higher therapeutic response. Moreover, both 6-TGN and 6-MeMPN levels were correlated with leucopenia. In AIH children, a positive correlation between azathioprine dosage and metabolite levels was also observed. The wide variability in thiopurine metabolites and therapeutic response in both IBD and AIH children was confirmed, pointing to the important role of treatment individualization. Monitoring of thiopurine metabolites combined with hematological tests, TPMT activity and clinical evaluation may be of interest for optimizing thiopurine therapy and minimizing toxicity in IBD and AIH children

The diploma thesis is devoted to complications of enteral nutrition in patients with idiopathic inflammatory bowel diseases. The incidence of these diseases has increased in recent years. Nutritional support is an important part of the treatment and the possibility of improving the nutritional status of patients, which plays an important role in responding to other forms of treatment itself or in pre-operative preparation. All forms of artificial nutrition might also cause certain complications. The subject of this work is to observe the complications of enteral nutrition, their solutions and the possibilities of their prevention.

Os mosquitos representam o grupo mais importante de vetores de doenças infecciosas para o homem. Para que possam ter sucesso no repasto sanguíneo e sejam capazes de adquirir os nutrientes necessários para a maturação dos ovos, a saliva desses vetores possui atividades anti-hemostáticas e imunomoduladoras. Sabendo-se da importância da saliva dos artrópodes hematófagos para sua alimentação e para a transmissão de patógenos causadores de doenças o objetivo deste estudo foi caracterizar as atividades imunomoduladoras de um peptídeo descrito no salivoma de A. aegypti, presente somente em glândulas salivares de mosquitos fêmeas e cujo papel biológico ainda é desconhecido. Observou-se que o peptídeo denominado AeMOPE-1 inibiu a produção de mediadores pró-inflamatórios por macrófagos ativados, assim como induziu a resposta imune para o perfil Th2. O tratamento com o AeMOPE-1 reduziu os sintomas clínicos da colite experimental. Essa é a primeira descrição de um peptídeo salivar de A. aegypti com atividade imunomoduladora em macrófagos e com potencial ação terapêutica. / Mosquitoes are the most important vectors of pathogens that causing infectious to humans. To female succeed in their hematophagous habit and thus acquire the nutrients necessary for the maturation of their eggs, their saliva has components with anti-hemostatic and immunomodulatory activities. Thus, the aim of this project was to study the role of a novel salivary peptide described in the A. aegypti sialome, present only in female salivay glands, whose biological functions are still unknown. It was observed that the peptide named AeMOPE-1 inhibited the production of pro-inflammatory mediators by activated macrophages, as well as induced the immune response to the Th2 profile. Treatment with AeMOPE-1 reduced the clinical symptoms of experimental colitis. This works presents the first salivary peptide from A. aegypti with potent activity on macrophage biology and potential clinical application.