Valutazione dell'efficacia e della sicurezza dell'Infliximab e della Ciclosporina Orale Microemulsione (Neoral) nei pazienti con colite ulcerosa severa refrattari agli steroidi per via endovenosa. Risultati preliminari di uno studio randomizzato controllato

Background. Intravenous steroids represent the mainstay of therapy for severe attacks of Ulcerative Colitis (UC). In steroid refractory patients, both iv cyclosporine (CsA) and infliximab (IFX) are valid rescue therapies. Several studies have shown that oral microemulsion CsA (Neoral) is equivalent to iv CsA in term of safety and efficacy in UC patients. Aim. To investigate the efficacy and safety of oral microemulsion CsA vs IFX in patients with severe attack of UC, refractory to iv steroids. Material and methods. From May 2006, all consecutive pts admitted for severe UC were considered eligible. Pts were treated with iv steroid, according to the Oxford regime. After 1 week of intensive treatment, pts non responder to the therapy and not candidate to the surgery, were asked to participate to the trial. They were randomised to receive IFX 5 mg/kg or oral CsA 5 mg/kg. Results. A total of 30 patients were randomised, 17 in the IFX group and 13 in the CsA group. One month after study inclusion, 9 patients of the IFX group (53%) and 7 pts of the CsA group (54%) were in clinical remission (p=0.96), with a Powell-Tuck index ≤ 3. At the end of the follow-up, 7 pts in the IFX group (41%) vs 4 in the CsA group (31%) (p=0.35) underwent colectomy. The total cost of the IFX therapy with IFX was 8.052,84 € versus 1.106,82 €, for each patient. Conclusions. Oral microemulsion CsA and IFX seem to be equivalent in term of efficacy and safety in severe UC patients refractory to iv steroids. In patients treated with IFX the cost of therapy were significantly higher.

Role of immunosuppressive and biologic therapy in pediatric IBD

We aimed to evaluate the role of anti-TNF-alpha therapy with infliximab and adalimumab in a cohort of pediatric patients followed by our Center from 2002 to 2012. The cohort of patients examined consisted of 40 patients: 34 with Crohn disease (85%), 5 with ulcerative colitis (12.5%), one with chronic pouchitis after IPAA for ulcerative colitis (2.5%). All patients were treated with the anti-TNF-α biologic agents infliximab and adalimumab. Thirty-six received infliximab therapy: 19/36 received only infliximab, 17/36 received infliximab and then adalimumab due to loss of response to infliximab and steroid dependency; 4 patients received only adalimumab (infliximab-naïve). Anti-TNF treatment was started before 18 years of age in 34 patients: 29 received infliximab and 5 started adalimumab during childhood. Medical charts were reviewed and safety and efficacy of anti-TNF-alpha have been determined in this population.

Development of new therapeutic approaches in the treatments of Inflammatory Bowel Diseases: functional food and nutraceuticals vs synthetic peptides based vaccines

Inflammatory Bowel Diseases (IBD) are intestinal chronic relapsing diseases which ethiopathogenesis remains uncertain. Several group have attempted to study the role of factors involved such as genetic susceptibility, environmental factors such as smoke, diet, sex, immunological factors as well as the microbioma. None of the treatments available satisfy several criteria at the same time such as safety, long-term remission, histopatological healing, and specificity. We used two different approaches for the development of new therapeutic treatment for Inflammatory Bowel Disease. The first is focused on the understanding of the potential role of functional food and nutraceuticals nutrients in the treatment of IBD. To do so, we investigated the role of Curcuma longa in the treatment of chemical induced colitis in mice model. Since Curcma Longa has been investigated for its antinflammatory role related to the TNFα pathway as well investigators have reported few cases of patients with ulcerative colites treated with this herbs, we harbored the hypothesis of a role of Curcuma Longa in the treatment f IBD as well as we decided to assess its role in intestinal motility. The second part is based on an immunological approach to develop new drugs to induce suppression in Crohn’s disease or to induce mucosa immunity such as in colonrectal tumor. The main idea behind this approach is that we could manipulate relevant cell-cell interactions using synthetic peptides. We demonstrated the role of the unique interaction between molecules expressed on intestinal epithelial cells such as CD1d and CEACAM5 and on CD8+ T cells. In normal condition this interaction has a role for the expansion of the suppressor CD8+ T cells. Here, we characterized this interaction, we defined which are the epitope involved in the binding and we attempted to develop synthetic peptides from the N domain of CEACAM5 in order to manipulate it.

Decorso delle coliti severe: un confronto tra colite ulcerosa e di Crohn

Pochi sono i dati disponibili sul decorso clinico della malattia di Crohn del colon severa(CD). L'obiettivo è quello di descrivere il decorso clinico della colite di Crohn severa (CC) in una coorte di pazienti isolata con CD del colon o ileocolica, e di confrontarlo con il decorso clinico di pazienti affetti da colite ulcerosa severa (UC). 34 pazienti con CC severa sono stati identificati retrospettivamente nella nostra coorte di 593 pazienti ricoverati (2003-2012) attraverso la valutazione di CDAI score e HBI. 169 pazienti con UC severa sono stati identificati retrospettivamente in una coorte di 449 pazienti ricoverati (2003-2012) attraverso la valutazione del score di Lichtiger e di Truelove-Witts. Abbiamo valutato questi risultati: risposta agli steroidi, risposta ai farmaci biologici, tasso di colectomia acuta, tasso di colectomia durante il follow-up, megacolon e tasso di infezione da citomegalovirus. Non abbiamo trovato differenze significative nella risposta agli steroidi e biologici, della percentuale di infezione da citomegalovirus e di megacolon, mentre il tasso di colectomia in acuto è risultato essere maggiore nei pazienti con CC rispetto ai pazienti con UC; anche la differenza tra i tassi di colectomia alla fine del follow-up è risultata non significativa. Con l'analisi univariata la giovane età alla diagnosi è associata ad un aumentato rischio di colectomia in assoluto (p = 0,024) e in elezione (p = 0.022), ma non in acuto. Il tasso globale di colectomia nei pazienti con CC severa è superiore a quella dei pazienti con UC severa , ma questo dato non è supportato da una diversa risposta clinica alla terapia steroidea o terapia di salvataggio con biologici. Il vero decorso clinico della colite di Crohn severa necessita di essere chiarito da studi prospettici che includano un numero maggiore di pazienti con questo sottogruppo di malattia.

Rilievi anatomo-patologici e batteriologici condotti sull’apparato gastroenterico di una metapopolazione di cinghiali (Sus scrofa)

Nell’ambito della patologia gastroenterica del suino sono comprese alcune malattie sostenute da batteri spirillari gram negativi, di cui sono disponibili numerose trattazioni riguardanti, soprattutto, l'aspetto epidemiologico e patogenetico. Per alcuni di questi agenti microbici, e per le relative manifestazioni patologiche, poco si conosce nel cinghiale selvatico, animale correlato filogeneticamente al suino domestico, ma compreso in un’ecologia completamente differente. Da queste premesse è nato un approccio di ricerca e studio del comportamento di questi microrganismi in una metapopolazione di cinghiali, abbattuti durante il piano di controllo della popolazione densità-dipendente nel Parco dei Gessi e Calanchi dell’Abbadessa (BO), cercando di rapportare le conoscenze riportate in letteratura sul suino domestico con quanto è scaturito dalle indagini condotte sul cinghiale selvatico. In particolare è stata indagata con metodica immunoistochimica la presenza di Lawsonia intracellularis, patogeno del suino responsabile di Enterite Proliferativa (EP), in secondo luogo sono state condotte indagini batteriologiche e istologiche da stomaco e intestino, finalizzate all’isolamento di microrganismi spirillari dei generi Campylobacter e Helicobacter, da correlare all’eventuale presenza di lesioni infiammatorie e ulcerative gastriche o enteriche valutate secondo sistemi a punteggio ottenuti dalla bibliografia o realizzati in base alla tipologia di infiltrato cellulare e alla sua localizzazione. In ultimo, a fini comparativi con uno studio condotto nel 2002-2004 nello steso Parco Regionale, sono stati monitorati i livelli di antibioticoresistenza di indicatori fecali usando metodiche internazionali standardizzate (Escherichia coli e Enterococcus faecium.) nonché su un numero significativo di isolati di Campylobacter lanienae, per ottenere indicazioni preliminari sull’andamento nei 10 anni trascorsi dello stato di inquinamento da farmaco del Parco stesso. I risultati ottenuti permettono di ampliare le conoscenze sulla flora enterica del cinghiale selvatico e pongono questioni di sicurezza pubblica sulla gestione dei mammiferi selvatici.

Ruolo della Interleuchina-6 nella colite ulcerosa long-standing: regolazione della p53 ed induzione delle cellule mesenchimali epiteliali

L’infiammazione cronica è un fattore di rischio di insorgenza del cancro, e la citochina infiammatoria IL-6 gioca un ruolo importante nella tumorigenesi. In questo studio abbiamo dimostrato che L’IL-6 down-regola l'espressione e l'attività di p53. In linee cellulari umane, IL-6 stimola la trascrizione dell’rRNA mediante espressione della proteina c-myc a livello post-trascrizionale in un meccanismo p38MAPK-dipendente. L'up-regolazione della biogenesi ribosomiale riduce l'espressione di p53 attraverso l'attivazione della via della proteina ribosomale-MDM2. La down-regolazione di p53 produce l’acquisizione di modifiche fenotipiche e funzionali caratteristiche della epitelio mesenchimale di transizione, un processo associato a trasformazione maligna e progressione tumorale. I nostri dati mostrano che questi cambiamenti avvengono anche nelle cellule epiteliali del colon di pazienti affetti da colite ulcerosa, un esempio rappresentativo di una infiammazione cronica soggetta a trasformazione neoplastica, che scompaiono dopo trattamento con farmaci antinfiammatori. Questi risultati svelano un nuovo effetto oncogenico indotto dall’IL-6 che può contribuire notevolmente ad aumentare il rischio di sviluppare il cancro non solo in pazienti con infiammazioni croniche, ma anche in quei pazienti con condizioni patologiche caratterizzate da elevato livello di IL-6 nel plasma, quali l'obesità e e il diabete mellito di tipo 2.

Expressionsunterschiede verschiedener miRNAs bei chronisch-entzündlichen Darmerkrankungen

Chronisch-entzündliche Darmerkrankungen konfrontieren unsere heutige Gesellschaft mit hohen Inzidenzraten in der westlichen Welt und zunehmend steigenden Inzidenzraten im asiatischen Raum. Die Folgen für die Patienten sind eine starke Beeinträchtigung der Lebensqualität, mit sozialen und wirtschaftlichen Folgen sowie ein erhöhtes Risiko für die Entwicklung kolorektaler Karzinome. Durch die Entdeckung von 22 nt langen, regulierenden RNAs, auch genannt miRNAs, wurde ein neuer Baustein im Verständnis zellulärer Regelprozesse und der Differenzierung und Aktivierung von Antworten etwa des Immunsystems entdeckt. Somit stellt sich die Frage nach der Bedeutung von miRNAs im Rahmen von chronisch-entzündlichen Darmerkrankungen. Hierzu wurden in dieser Arbeit über ein miRNA-Array System 12 miRNAs als potentiell relevante Ziele identifiziert und an einem Kollektiv aus insgesamt 131 Patienten und 163 Biopsien aus dem Bereich des Darmes überprüft. Es zeigte sich hierbei, dass im Rahmen eines Morbus Crohn mit Befall des Dickdarms die miRNAs let-7d und miR-22 in gesteigerter Expression vorlagen. Da im terminalen Ileum eine gesonderte Immunsituation vorliegt, wurde dieser Bereich zusätzlich bei der Erkrankung Morbus Crohn untersucht. Es zeigten sich Expressionsveränderungen für die miRNAs miR-30e, miR-185, miR-374b und miR-424. Bei Patienten mit einer Colitis ulcerosa waren die miRNAs let-7d, miR-185 und miR-424 in ihrem Expressionsverhalten verändert. Zusätzlich konnte gezeigt werden, dass in Abhängigkeit vom Entzündungsgrad bei bestehender Colitis ulcerosa eine zunehmenden Überexpression der miRNAs let-7d, miR-185 und miR-424 erfolgte. Die miRNAs miR-18a und miR-185 wiesen unter Remissionsbedingungen Expressionsveränderungen auf und lassen somit den Verdacht eines protektiven Effektes aufkommen. Mit Hilfe von computerbasierten Datenbankanalysen konnten gemeinsam regulierenden miRNAs Proteine und Pathways zugeordnet werden, welche einen Zusammenhang mit bereits pathogenetisch bestätigten Signalwegen wie etwa dem nF-ĸB und MAPK-Signalweg nahelegen. Auch konnte herausgearbeitet werden, dass einige, der von diesen miRNAs regulierten Proteine, bereits in veröffentlichten Arbeiten als fehlreguliert festgestellt wurden, jedoch blieb die Ursache dieser Fehlregulation gänzlich unbekannt. Mit den in dieser Arbeit erhobenen Daten konnte gezeigt werden, dass eine Kongruenz der Befunde vorliegt, welche einen Zusammenhang der miRNA-Expression mit der Fehlregulation bestimmter Proteine nicht nur nahelegt, sondern darüber hinaus auch noch einige weitere potentielle Proteinziele für weitere Untersuchungen aufführt. Dazu ist es jedoch notwendig, die Relevanz der hier entdeckten, computerbasierten Proteine in zukünftigen Untersuchungen einer genauen Prüfung zu unterziehen.

Messung von 6-Thioguanosin-5'-Monophosphat, -Diphosphat und -Triphosphat in Erythrozyten und Ansprechen auf eine Azathioprin-Therapie in chronisch entzündlichen Darmerkrankungen

Die Ätiopathogenese von Morbus Crohn und Colitis ulcerosa ist bis heute unklar. Azathioprin ist das wichtigste Immunsuppressivum in der Therapie der beiden Erkrankungen. Der Wirkmechanismus ist unklar. Eine Entschlüsselung des Wirkmechanismus könnte zu einer Optimierung des Medikamentes mit Reduktion der unerwünschten Wirkungen genutzt werden. Der Metabolismus von Azathioprin ist komplex. TGTP löst in Lamina propria-T-Zellen Apoptose aus und wird als aktiver Metabolit betrachtet. In einer Stichprobe von 133 Patienten konnte gezeigt werden, dass hohe TGTP-Spiegel, insbesondere in Verbindung mit niedrigen TGDP-Spiegeln, ein Ansprechen auf die Therapie prognostizieren können.

Methylierungs- und Expressionsuntersuchungen zur Aufklärung der molekularen Pathogenese in chronisch entzündlichen Darmerkrankungen

Unter der Bezeichnung Chronisch Entzündliche Darmerkrankungen (CED) werden zwei Erscheinungsformen, Colitis Ulcerosa (CU) und Morbus Crohn (MC) zusammengefasst. Das Leitsymptom von CED sind chronische Entzündungen des Magen-Darm-Trakts, insbesondere des terminalen Ileum und des Colons. Es wird angenommen, dass eine aberrante Immunantwort auf das intestinale Mikrobiom in einem genetisch prädisponierten Individuum zur Entstehung von CED führt.rnFür diese Studie ist der genetische, bzw. epigenetische Aspekt, der Pathogenese von CU und MC von besonderem Interesse. In verschiedenen Assoziationsstudien wurden bereits 163 mit CED assoziierte, krankheitsrelevante Gen Loci identifiziert. Zusätzlich wurden Studien durchgeführt, die Methylierungs- und Expressionsunterschiede in Gewebe oder Blut von CED-Patienten gegenüber gesunden Probanden (Kontrollen) aufzeigten. rnIn der vorliegenden Studie wurden entzündliche- und nicht-entzündliche Gewebeproben von CU- (Colon) und MC-Patienten (terminales Ileum und Colon) und gesunden Probanden (terminales Ileum und Colon; nicht entzündlich) auf genspezifischer- und genomweiter Ebene auf Methylierungs- und Expressionsunterschiede hin untersucht. Im Rahmen der genspezifischen Analysen wurde in vier Genen (IL17REL, MUC2, MUC6, MUC15) eine aberrante Methylierung im Vergleich der MC- oder CU-Gewebeproben mit den Kontrollen detektiert. Die an 24 ausgewählten CU Colon-Proben (NE und E) und Colon Kontrollen durchgeführte genomweite Methylierungsanalyse zeigte aberrante Methylierungsmuster in über 2500 Genen im Vergleich der entzündlichen CU Colon E-Proben mit den Kontrollen. Fünf dieser Gene (BACH2, STAT3, STAT4, STK4 und WIPF1) wurden ausgewählt und die Veränderung der Methylierung an einem größeren Patientenkollektiv, welches auch Proben von MC-Patienten umfasst, bestätigt. Zusätzlich zu der aberranten Methylierung wurden Expressionsveränderungen des IL17REL-, MUC6- und STAT4-Gens in MC-Patienten sowie des MUC2-Gens in CU-Patienten identifiziert. rnDa über die Promoterregion und Funktion von IL17REL nur sehr wenig bis gar nichts bekannt ist, wurden zusätzlich Promoteranalysen mittels Dual-Luciferase-Assay durchgeführt. Die Ergebnisse zeigten, dass die höchste Aktivität des putativen IL17REL-Promoters im Bereich -806 – -8 vor der 5’UTR zu finden ist. In diesem Bereich lagen auch die in der Methylierungsanalyse untersuchten CpGs.rn

Reduced muscle mass and bone size in pediatric patients with inflammatory bowel disease

BACKGROUND: Decreased bone mineral density has been reported in children with inflammatory bowel disease (IBD). We used peripheral quantitative computed tomography (pQCT) to assess bone mineralization, geometry, and muscle cross-sectional area (CSA) in pediatric IBD. METHODS: In a cross-sectional study, pQCT of the forearm was applied in 143 IBD patients (mean age 13.9 +/- 3.5 years); 29% were newly diagnosed, 98 had Crohn's disease, and 45 had ulcerative colitis. Auxological data, cumulative glucocorticoid dose, disease activity indices, laboratory markers for inflammation, and bone metabolism were related to the results of pQCT. RESULTS: Patients were compromised in height (-0.82 +/- 1.1 SD), weight (-0.77 +/- 1.0 SD), muscle mass (-1.12 +/- 1.0 SD), and total bone cross-sectional area (-0.79 +/- 1.0 SD) compared to age- and sex-matched healthy controls (z-scores). In newly diagnosed patients, the ratio of bone mineral mass per muscle CSA was higher than in those with longer disease duration (1.00 versus 0.30, P = 0.007). Serum albumin level and disease activity correlated with muscle mass, accounting for 41.0% of variability in muscle mass (P < 0.01). The trabecular bone mineral density z-score was on average at the lower normal level (-0.40 +/- 1.3 SD, P < 0.05). CONCLUSIONS: Reduced bone geometry was explained only in part by reduced height. Bone disease in children with IBD seems to be secondary to muscle wasting, which is already present at diagnosis. With longer disease duration, bone adapts to the lower muscle CSA. Serum albumin concentration is a good marker for muscle wasting and abnormal bone development.

Changes in CXCL12/CXCR4-chemokine expression during onset of colorectal malignancies

Chemokines have been proposed to contribute to tumour growth and metastatic spread of several cancer entities. Here, we examined the relative levels of CXCL12/CXCR4 in resection specimens from patients with different malignant and non-malignant colorectal diseases as well as colorectal liver metastases (CRLM). CXCL12/CXCR4 mRNA and protein expression profiles were assessed by quantitative real-time PCR, Western blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry in resection specimens from patients with ulcerative colitis (UC; n = 15), colorectal adenoma (CRA; n = 15), colorectal adenocarcinoma (CRC; n = 47) and CRLM (n = 16). Corresponding non-affected tissues served as control. In contrast to UC tissues, CXCL12 showed a distinct down-regulation in CRA, CRC and CRLM specimens, whereas the corresponding receptor CXCR4 demonstrated a significant up-regulation in CRC and CRLM related to corresponding non-affected tissues (p < 0.05, respectively). Our results strongly suggest an association between CXCL12/CXCR4 expression and the induction of CRA, CRC and the development of CRLM. Therefore, CXCR4 may be a potential target for specific therapeutic interventions.

Epidemiology of inflammatory bowel disease: Is there a shift towards onset at a younger age?

Increasing numbers of paediatric and adolescent patients with Crohn disease (CD) and ulcerative colitis (UC) are reported. To determine whether this observation is a consequence of a shift towards onset at a younger age, we analysed retrospective data from patients enrolled in the Swiss IBD Cohort Study (SIBDCS).

Genetic evidence supporting the association of protease and protease inhibitor genes with inflammatory bowel disease: a systematic review

As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs) in inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC]) to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors) were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD) on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH), dystroglycan (DAG1), macrophage-stimulating protein (MST1) and ubiquitin-specific peptidase 4 (USP4), all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4 protease inhibitors), with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family.

Association of a common vitamin D-binding protein polymorphism with inflammatory bowel disease

Inflammatory bowel diseases (IBDs), Crohn's disease, and ulcerative colitis (UC), are multifactorial disorders, characterized by chronic inflammation of the intestine. A number of genetic components have been proposed to contribute to IBD pathogenesis. In this case-control study, we investigated the association between two common vitamin D-binding protein (DBP) genetic variants and IBD susceptibility. These two single nucleotide polymorphisms (SNPs) in exon 11 of the DBP gene, at codons 416 (GAT>GAG; Asp>Glu) and 420 (ACG>AAG; Thr>Lys), have been previously suggested to play roles in the etiology of other autoimmune diseases.

Deep resequencing of GWAS loci identifies independent rare variants associated with inflammatory bowel disease

More than 1,000 susceptibility loci have been identified through genome-wide association studies (GWAS) of common variants; however, the specific genes and full allelic spectrum of causal variants underlying these findings have not yet been defined. Here we used pooled next-generation sequencing to study 56 genes from regions associated with Crohn's disease in 350 cases and 350 controls. Through follow-up genotyping of 70 rare and low-frequency protein-altering variants in nine independent case-control series (16,054 Crohn's disease cases, 12,153 ulcerative colitis cases and 17,575 healthy controls), we identified four additional independent risk factors in NOD2, two additional protective variants in IL23R, a highly significant association with a protective splice variant in CARD9 (P < 1 × 10(-16), odds ratio ≈ 0.29) and additional associations with coding variants in IL18RAP, CUL2, C1orf106, PTPN22 and MUC19. We extend the results of successful GWAS by identifying new, rare and probably functional variants that could aid functional experiments and predictive models.