Modulation of inflammatory mediators by Opuntia ficus-indica and Prunus avium bioproducts using an in vitro cell-based model of intestinal inflammation

Dissertation to obtain a Master Degree in Biotechnology

Homogneous time resolved fluorescence: a new technique for detection and quantifications of infliximab in autoimmune patients

RESUMO:As terapias biológicas revolucionaram o tratamento das doenças autoimunes nos últimos anos. Tipicamente têm como alvos mediadores importantes no mecanismo das doenças. Os antagonistas do fator de necrose tumoral-α (TNF-α) são um grupo de agentes biológicos muito prescrito, pois estão indicados no tratamento de doenças imuno-mediadas comuns, tais como artrite reumatoide, artrite idiopática juvenil, artrite psoriática, espondilite anquilosante, doença de Crohn e colite ulcerosa. Com o uso frequente de inibidores do TNF-α, tem-se tornado evidente que estes agentes têm um potencial imunogénico importante, que pode comprometer o prognóstico a longo prazo dos doentes cronicamente tratados. A produção de anticorpos anti-fármaco parece causar falência terapêutica secundária em muitos doentes. Um dos efeitos dos anticorpos anti-fármaco é o aumento da eliminação do fármaco. A eliminação do fármaco, por sua vez, varia entre indivíduos, refletindo diferentes perfis farmacocinéticos. A determinação dos níveis séricos mínimos do agente anti-TNF-α é assim muito informativa e pode auxiliar nas decisões terapêuticas. Contudo, os testes imunológicos para determinar as concentrações séricas do fármaco não estão facilmente disponíveis na prática clínica. De forma a investigar uma nova técnica potencialmente fidedigna e prática para a deteção e quantificação dos agentes biológicos anti-TNF-α, foi testada a técnica por HTRF (homogeneous time-resolved fluorescence resonance energy transfer) para a determinação de concentrações séricas de infliximab. Apesar de apresentar algumas limitações relacionadas com as condições de leitura da fluorescência, esta técnica provou obter resultados próximos das concentrações obtidas por ELISA (enzyme-linked immunosorbent assay) bridging. Adicionalmente, tem a vantagem de ser de execução muito mais fácil e rápida. Deste modo, a técnica por HTRF poderá ser otimizada e tornar-se uma valiosa ferramenta laboratorial para orientar as decisões terapêuticas em doentes autoimunes com falência da terapêutica anti-TNF-α.--------- ABSTRACT: Biologic therapies revolutionized the treatment of autoimmune diseases in the last years. Typically, they target important disease mediators. Tumor necrosis factor-alpha (TNF-α) antagonists constitute a very prescribed group of biologic agents as they are indicated for the treatment of common immune-mediated diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease and ulcerative colitis. With the increasing use of TNF-α inhibitors it has been noticed that they have an important immunogenic potential that can compromise long-term outcomes in chronically treated patients. The production of anti-drug antibodies seems to cause secondary therapeutic failure in many patients. One of the effects of anti-drug antibodies is the enhancement of drug clearance. Drug clearance, in turn, varies among individuals, reflecting different pharmacokinetic profiles. Determination of serum anti-TNF-α drug trough levels is though very informative and could support treatment decisions. However, immunologic assays to determine drug serum concentrations are not readily available in clinical practice. In order to investigate a potentially reliable and practical new technique for detection and quantification of anti-TNF-α biologic agents, homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technique was tested for determination of serum infliximab concentrations. Although presenting some limitations related with fluorescence reading conditions, this technique proved to give results close to the concentrations obtained by the widely used bridging enzyme-linked immunosorbent assay (ELISA). In addition, it has the advantage of being much easier and faster to perform. Thus, HTRF technique can be optimized and become a valuable laboratorial tool to guide treatment decisions in autoimmune patients with anti-TNF-α therapy failure.

Pelvic lymphoscintigraphy: contribution to the preoperative staging of rectal cancer

PURPOSE: Preservation of the anal sphincter in surgery for cancer of the distal rectum in an attempt to avoid colostomy has been a main concern of colorectal surgeons. Various proposed procedures contradict oncological principles, especially with respect to pelvic lymphadenectomy. Therefore, prior knowledge of pelvic lymph node involvement is an important factor in choosing the operative technique, i.e., radical or conservative resection. Introduction of ultrasound, computerized tomography, and magnetic resonance have made preoperative study of the area possible. Nevertheless, these resources offer information of an anatomical nature only. Lymphoscintigraphy enables the morphological and functional evaluation of the pelvic area and contributes toward complementing the data obtained with the other imaging techniques. The objective of this prospective study is twofold: to standardize the lymphoscintigraphy technique and to use it to differentiate patients with rectal cancer from those with other coloproctologic diseases. CASUISTIC AND METHODS: Sixty patients with various coloproctologic diseases were studied prospectively. Ages ranged from 21 to 96 years (average, 51 and median, 55 years). Twenty-six patients were male and 34 were female. Thirty patients had carcinoma of the distal rectum as diagnosed by proctologic and anatomic-pathologic examinations, 20 patients had hemorrhoids, 5 had chagasic megacolon, 2 had diverticular disease, 2 had neoplasm of the right colon, and 1 had ulcerative colitis as diagnosed by proctologic exam and/or enema. The lymphoscintigraphy method consisted of injecting 0.25 mL of a dextran solution marked with radioactive technetium-99m into the right and left sides of the perianal region and obtaining images with a gamma camera. The results were analyzed statistically with a confidence level of 95% (P < .05) using the following statistical techniques: arithmetic and medium average, Fisher exact test, chi-square test corrected for continuity according to Yates, and distribution tables for the number of patients. RESULTS: In rectal cancer, the tracer progresses unilaterally or is absent; in other patients, the progress of the tracer is bilateral and symmetrical, although its progress may be slow. Statistical tests showed with high significance that the agreement index between the clinical diagnosis and the result of the lymphoscintigraphic exam was 93%. CONCLUSIONS: Lymphoscintigraphy is a standardized, painless, and harmless test that can be performed in all cases; it differentiates patients with rectal cancer from those with other coloproctological diseases.

Inflammatory bowel diseases: principles of nutritional therapy

Inflammatory Bowel Diseases - ulcerative colitis and Crohn's disease- are chronic gastrointestinal inflammatory diseases of unknown etiology. Decreased oral intake, malabsorption, accelerated nutrient losses, increased requirements, and drug-nutrient interactions cause nutritional and functional deficiencies that require proper correction by nutritional therapy. The goals of the different forms of nutritional therapy are to correct nutritional disturbances and to modulate inflammatory response, thus influencing disease activity. Total parenteral nutrition has been used to correct and to prevent nutritional disturbances and to promote bowel rest during active disease, mainly in cases of digestive fistulae with high output. Its use should be reserved for patients who cannot tolerate enteral nutrition. Enteral nutrition is effective in inducing clinical remission in adults and promoting growth in children. Due to its low complication rate and lower costs, enteral nutrition should be preferred over total parenteral nutrition whenever possible. Both present equal effectiveness in primary therapy for remission of active Crohn's disease. Nutritional intervention may improve outcome in certain individuals; however, because of the costs and complications of such therapy, careful selection is warranted, especially in patients presumed to need total parenteral nutrition. Recent research has focused on the use of nutrients as primary treatment agents. Immunonutrition is an important therapeutic alternative in the management of inflammatory bowel diseases, modulating the inflammation and changing the eicosanoid synthesis profile. However, beneficial reported effects have yet to be translated into the clinical practice. The real efficacy of these and other nutrients (glutamine, short-chain fatty acids, antioxidants) still need further evaluation through prospective and randomized trials.

Extensive subcutaneous emphysema as a presentation of ischemic colitis

Introduction . Subcutaneous emphysema is usually benign and self-limited; however, it may be associated with a life-threating situation. Case Report . An elderly woman with progressive malaise with extensive subcutaneous emphysema (cervical to abdominal wall) was observed at the emergency department. Colonic perforation was diagnosed and the patient underwent surgery. Intraoperatively, necrosis and perforation of the sigmoid colon into the retroperitoneum were found and a Hartmann procedure was performed. Conclusion . Cervical and thoracic subcutaneous emphysema may be the first sign of intra-abdominal lesion.

Verstärkung der suppressiven Eigenschaften von regulatorischen T-Zellen durch den Alanyl-Aminopeptidase-Inhibitor Phebestin in vitro sowie im DSS-Colitis Modell in vivo

Regulatorische T-Zellen; Treg; Colitis ulcerosa; DSS; CED; APN; Phebestin

Untersuchung der Rolle von Chk1 in der entzündungsbasierten kolorektalen Karzinogenese anhand eines zellulären Colitis-Modells

Otto-von-Guericke-Universität Magdeburg, Fakultät für Naturwissenschaften, Dissertation, 2016

Prevalence and clinical importance of mesenteric venous thrombosis in the swiss inflammatory bowel disease cohort.

OBJECTIVE. The purpose of this study was to evaluate the prevalence of mesenteric venous thrombosis (MVT) in the Swiss Inflammatory Bowel Disease Cohort Study and to correlate MVT with clinical outcome. MATERIALS AND METHODS. Abdominal portal phase CT was used to examine patients with inflammatory bowel disease (IBD). Two experienced abdominal radiologists retrospectively analyzed the images, focusing on the superior and inferior mesenteric vein branches and looking for signs of acute or chronic thrombosis. The location of abnormalities was registered. The presence of MVT was correlated with IBD-related radiologic signs and complications. RESULTS. The cases of 160 patients with IBD (89 women, 71 men; Crohn disease [CD], 121 patients; ulcerative colitis [UC], 39 patients; median age at diagnosis, 27 years for patients with CD, 32 years for patients with UC) were analyzed. MVT was detected in 43 patients with IBD (26.8%). One of these patients had acute MVT; 38, chronic MVT; and four, both. The prevalence of MVT did not differ between CD (35/121 [28.9%]) and UC (8/39 [20.5%]) (p = 0.303). The location of thrombosis was different between CD and UC (CD, jejunal or ileal veins only [p = 0.005]; UC, rectocolic veins only [p = 0.001]). Almost all (41/43) cases of thrombosis were peripheral. MVT in CD patients was more frequently associated with bowel wall thickening (p = 0.013), mesenteric fat hypertrophy (p = 0.005), ascites (p = 0.002), and mesenteric lymph node enlargement (p = 0.036) and was associated with higher rate of bowel stenosis (p < 0.001) and more intestinal IBD-related surgery (p = 0.016) in the outcome. Statistical analyses for patients with UC were not relevant because of the limited population (n = 8). CONCLUSION. MVT is frequently found in patients with IBD. Among patients with CD, MVT is associated with bowel stenosis and CD-related intestinal surgery.

Periodontitis and gingivitis in inflammatory bowel disease: a case-control study.

BACKGROUND: The oral cavity is frequently affected in patients with inflammatory bowel disease (IBD), especially in patients with Crohn's disease (CD). Periodontitis is thought to influence systemic autoimmune or inflammatory diseases. We aimed to analyze the relationship of periodontitis and gingivitis markers with specific disease characteristics in patients with IBD and to compare these data with healthy controls. METHODS: In a prospective 8-month study, systematic oral examinations were performed in 113 patients with IBD, including 69 patients with CD and 44 patients with ulcerative colitis. For all patients, a structured personal history was taken. One hundred thirteen healthy volunteers served as a control group. Oral examination focussed on established oral health markers for periodontitis (bleeding on probing, loss of attachment, and periodontal pocket depth) and gingivitis (papilla bleeding index). Additionally, visible oral lesions were documented. RESULTS: Both gingivitis and periodontitis markers were higher in patients with IBD than in healthy control. In univariate analysis and logistic regression analysis, perianal disease was a risk factor for periodontitis. Nonsmoking decreased the risk of having periodontitis. No clear association was found between clinical activity and periodontitis in IBD. In only the CD subgroup, high clinical activity (Harvey-Bradshaw index > 10) was associated with 1 periodontitis marker, the loss of attachment at sites of maximal periodontal pocket depth. Oral lesions besides periodontitis and gingivitis were not common, but nevertheless observed in about 10% of patients with IBD. CONCLUSIONS: IBD, and especially perianal disease in CD, is associated with periodontitis. Optimal therapeutic strategies should probably focus on treating both local oral and systemic inflammation.

Proteomic analysis of cytokine induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases.

A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from (35)S-radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon-gamma, interleukin-1beta and interleukin-6. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in-gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl-tRNA synthetase, indoleamine-2,3-dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon-induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine-2,3-dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn's disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.

Prevalence and clinical importance of mesenteric venous thrombosis in the Swiss Inflammatory Bowel Disease Cohort

OBJECTIVE: The purpose of this study was to evaluate the prevalence of mesenteric venous thrombosis (MVT) in the Swiss Inflammatory Bowel Disease Cohort Study and to correlate MVT with clinical outcome. MATERIALS AND METHODS: Abdominal portal phase CT was used to examine patients with inflammatory bowel disease (IBD). Two experienced abdominal radiologists retrospectively analyzed the images, focusing on the superior and inferior mesenteric vein branches and looking for signs of acute or chronic thrombosis. The location of abnormalities was registered. The presence of MVT was correlated with IBD-related radiologic signs and complications. RESULTS: The cases of 160 patients with IBD (89 women, 71 men; Crohn disease [CD], 121 patients; ulcerative colitis [UC], 39 patients; median age at diagnosis, 27 years for patients with CD, 32 years for patients with UC) were analyzed. MVT was detected in 43 patients with IBD (26.8%). One of these patients had acute MVT; 38, chronic MVT; and four, both. The prevalence of MVT did not differ between CD (35/121 [28.9%]) and UC (8/39 [20.5%]) (p = 0.303). The location of thrombosis was different between CD and UC (CD, jejunal or ileal veins only [p = 0.005]; UC, rectocolic veins only [p = 0.001]). Almost all (41/43) cases of thrombosis were peripheral. MVT in CD patients was more frequently associated with bowel wall thickening (p = 0.013), mesenteric fat hypertrophy (p = 0.005), ascites (p = 0.002), and mesenteric lymph node enlargement (p = 0.036) and was associated with higher rate of bowel stenosis (p < 0.001) and more intestinal IBD-related surgery (p = 0.016) in the outcome. Statistical analyses for patients with UC were not relevant because of the limited population (n = 8). CONCLUSION: MVT is frequently found in patients with IBD. Among patients with CD, MVT is associated with bowel stenosis and CD-related intestinal surgery.

High altitude journeys and flights are associated with the increased risk of flares in IBD patients

Background: There is increasing evidence that hypoxia induces inflammation in the gastrointestinal tract. The clinical impact of hypoxia in patients with inflammatory bowel disease (IBD) is so far poorly investigated. Aim: We wanted to evaluate if flights and journeys to regions >= 2000 meter above sea level are associated with the occurrence of flares in IBD patients in the following 4 weeks. Methods: A questionnaire was completed by inpatients and outpatients of the IBD clinics of three tertiary referral centers presenting with an IBD flare. Patients were inquired about their habits in the 4 weeks prior to the flare. Patients with flares were matched with an IBD group in remission during the observation period (according to age, gender, smoking habits, and medication). Results: A total of 103 IBD patients were included (43 Crohn's disease (CD), whereof 65% female, 60 ulcerative colitis, whereof 47% female, mean age 39.3 ± 14.6 years for CD and 43.1 ± 14.2 years for UC). Fifty-two patients with flares were matched to 51 patients without flare. Overall, IBD-patients with flares had significantly more frequently a flight and/or journey to regions >= 2000 meters above sea level in the observation period compared to the patients in remission (21/52 (40.4%) vs. 8/51 (15.7%), p = 0.005). There was a statistically significant correlation between the occurrence of a flare and a flight and/or journey to regions >= 2000 meters above sea level among CD patients with flares as compared to CD patients in remission (8/21 (38.1%) vs. 2/22 (9.1%), p = 0.024). A trend for more frequent flights and high-altitude journeys was observed in UC patients with flares (13/31 (41.9%) vs. 6/29 (20.7%), p = 0.077). Mean flight duration was 5.8 ± 4.3 hours. The groups were controlled for the following factors (always flare group cited first): age (39.6 ± 13.4 vs. 43.5 ± 14.6, p = 0.102), smoking (16/52 vs. 10/51, p = 0.120), regular sports activities (32/52 vs. 33/51, p = 0.739), treatment with antibiotics in the 4 weeks before flare (8/52 vs. 7/51, p = 0.811), NSAID intake (12/52 vs. 7/51, p = 0.221), frequency of chronic obstructive pulmonary disease (both groups 0) and oxygen therapy (both groups 0). Conclusion: IBD patients with a flare had significantly more frequent flights and/or high-altitude journeys within four weeks prior to the IBD flare compared to the group that was in remission. We conclude that flights and stays in high altitude are a risk factor for IBD flares.

IRF4 regulates IL-17A promoter activity and controls RORγt-dependent Th17 colitis in vivo.

Background: The transcription factor IRF4 is involved in several T-cell-dependent chronic inflammatory diseases. To elucidate the mechanisms for pathological cytokine production in colitis, we addressed the role of the IRF transcription factors in human inflammatory bowel disease (IBD) and experimental colitis.Methods: IRF levels and cytokine production in IBD patients were studied as well as the effects of IRF4 deficiency in experimental colitis.Results: In contrast to IRF1, IRF5, and IRF8, IRF4 expression in IBD was augmented in the presence of active inflammation. Furthermore, IRF4 levels significantly correlated with IL-6 and IL-17 mRNA expression and to a lesser extent with IL-22 mRNA expression in IBD. To further explore the role of IRF4 under in vivo conditions, we studied IRF4-deficient and wildtype mice in experimental colitis. In contrast to DSS colitis, IRF4 deficiency was protective in T-cell-dependent transfer colitis associated with reduced ROR alpha/gamma t levels and impaired IL-6, IL-17a, and IL-22 production, suggesting that IRF4 acts as a master regulator of mucosal Th17 cell differentiation. Subsequent mechanistic studies using database analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays identified a novel IRF4 binding site in the IL-17 gene promoter. Overexpression of IRF4 using retroviral infection induced IL-17 production and IL-17 together with IL-6 induced ROR gamma t expression.Conclusions: IRF4 can directly bind to the IL-17 promotor and induces mucosal ROR gamma t levels and IL-17 gene expression thereby controlling Th17-dependent colitis. Targeting of this molecular mechanism may lead to novel therapeutic approaches in human IBD.

Anti-TNF Therapy in the Swiss Inflammatory Bowel Disease G8 Cohort

Background: A nti-TNF d rugs (Infliximab (IFX), Adalimumab (ADA), Certolizumab pegol (CZP)) are effective in inducing and maintaining response a nd remission in i nflammatory bowel disease (IBD). Insufficient response or side effects may lead to a switch o f the anti-TNF d rug. W e aimed to e valuate the frequency and reasons for anti-TNF switches. Methods: Analysis of data from the Swiss Inflammatory Bowel Disease Cohort (SIBDCS). Eighty percent of included patients were recruited in hospitals and 20% from private practice. Results: From 2,058 patients ( 1,172 with Crohn's disease (CD), 842 with ulcerative colitis (UC) and 44 with indeterminate colitis (IC)), 772 received at least one anti-TNF. Forty-eight % of patients w ith CD, 23% with U C, a nd 30% with IC w ere ever treated with an anti-TNF drug. There was no gender difference with respect to the frequency of a nti-TNF treatment. A total of 584 patients (76%) were treated with one, 142 (18%) with two, and 46 (6%) with three anti-TNF (of which 32 were female). A total of 89% patients were treated with IFX, 28% ADA and 13% with CZP. Overall response rate (defined as drop in CDAI >100 points) to anti-TNF was 50%, with best response rates for the first used anti-TNF. Reasons t o switch t he anti-TNF w ere in 11% a primary non-response, in 38% a loss of response and in 36% anti-TNF s ide effects o r intolerance ( reasons for 15% of treatment failures not documented). Conclusion: A nti-TNF d rugs were used in h alf of the CD patients a nd in o ne quarter of U C patients. Anti-TNF d rug switch d ue to insufficient response a nd/or side effects w as necessary in one quarter of IBD patients. IFX was mainly used as first-line therapy. Best response rates were observed for the first used anti-TNF. Following analyses will identify risk median treatment duration as well as risk factors for anti-TNF switch.

MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts.

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.