A dose escalating, placebo controlled, double blind, single dose and multidose, safety and tolerability study of fontolizumab, a humanised anti-interferon gamma antibody, in patients with moderate to severe Crohn's disease.

INTRODUCTION: This study was designed to evaluate the safety of fontolizumab, a humanised anti-interferon gamma antibody, in patients with moderate to severe Crohn's disease (CD). PATIENTS AND METHODS: Forty five patients with a CD activity index (CDAI) of 250-450 were randomised in a double blind, placebo controlled, dose escalating fashion to receive single doses of fontolizumab (0.1, 1.0, and 4.0 mg/kg) or placebo. By day 29, patients with clinical response were re-randomised to receive three additional doses of one half their initial fontolizumab dose or placebo at four weekly intervals. Primary objectives were safety and tolerability. Secondary outcomes included assessments of immunogenicity, clinical activity, and potential pharmacodynamic surrogates. RESULTS: Treatment was generally well tolerated. There were slightly more reports of chills, flu-like syndrome, asthenia, nausea, and vomiting in the 1.0 mg and 4.0 mg/kg fontolizumab cohorts. Two serious adverse events rated as worsening of CD occurred under fontolizumab. Antibodies to fontolizumab were confirmed in one patient. No differences in clinical activity parameters were noted between any of the active treatment groups and placebo, with the placebo group having a particularly favourable outcome (60% response and 40% remission). By day 29, a more enhanced decrease in median Crohn's disease endoscopic index of severity (p = 0.02) and serum C reactive protein (p<0.001) was observed in the 4.0 mg/kg (n = 14) fontolizumab cohort compared with placebo (n = 10). Pharmacodynamic effects were observed by immunohistochemistry. CONCLUSIONS: Fontolizumab was well tolerated with minimal immunogenicity at doses of up to 4.0 mg/kg in patients with CD. A biological activity of fontolizumab is suggested.

Abrogation of IFN-gamma mediated epithelial barrier disruption by serine protease inhibition.

The intestinal barrier function is often impaired in a variety of diseases including chronic inflammatory bowel disease. Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of the tight junction protein complex. IFN-gamma has been widely studied for its effect on barrier function and tight junction structures but its mode of action remains unclear. Since the claudin family of tight junction proteins is proposed to be involved in barrier maintenance we studied the effect of IFN-gamma on claudin expression in relation to epithelial barrier function. Cycloheximide and protease inhibitors were used to study mechanisms of IFN-gamma mediated barrier disruption. Intestinal epithelial cells were exposed to IFN-gamma and permeability was evaluated by horse radish peroxidase (HRP) and 4 kD FITC-dextran fluxes. Occludin and claudin-1, -2, -3, and -4 tight junction protein expression was determined by Western blotting. Occludin and claudin-2 protein expression was dramatically reduced after IFN-gamma exposure, which correlated with increased permeability for HRP and FITC-dextran. Interestingly, cleavage of claudin-2 was observed after incubation with IFN-gamma. Serine protease inhibitor AEBSF completely abrogated IFN-gamma mediated barrier disruption which was associated with preservation of claudin-2 expression. Moreover, IFN-gamma induced loss of barrier integrity was found to affect claudin-2 and occludin expression through different mechanisms. Since inhibition of serine protease activity abrogates IFN-gamma mediated barrier disruption this may be an important target for therapeutic intervention.

Report on the Dutch consensus development meeting for implementation and further development of population screening for colorectal cancer based on FOBT.

A consensus development meeting was held to evaluate whether or not in the Netherlands all requirements were fulfilled for implementation of population screening with FOBT for colorectal cancer, or whether consensus was present that fulfilment by additional research or organisational actions could be obtained within 2-3 years. There was consensus that all classical Wilson and Jungner (1968) criteria, and six additional ones added more recently, had already been fulfilled or could be fulfilled within 2-3 years. Consequently, it was concluded that a national population screening for colorectal cancer should be implemented and carried out in the Netherlands in line with current national and European cancer screening programmes. A list of organisational actions to be taken was established. Research that is needed before the actual national launch of the screening within 2-3 years has been defined. Priorities have to be set for research and organisational actions for the coming 2-3 years for the implementation of population screening. In addition, research suggestions have been defined for the next 10-15 years for evaluation and/or improvement of implemented FOBT screening, and for future screening methodology. It was considered essential that infrastructure for future research would be embedded in the screening programme. A project group to arrange this should be formed.

Endoscopic treatment of strictures in Crohn's disease.

Patients with Crohn's disease often develop (recurring) intestinal stenosis. This is a result of continuous activation of fibrogenic cells by ongoing inflammation. Surgery is usually needed and consists of intestinal resection or strictureplasty. Medical therapy has not proven to be successful. Over the years endoscopic treatment has become more important. Uncomplicated stenosis, with a maximal length of 4 cm, can be treated by balloon dilatation. Indications, procedure and results are discussed. More recently, local corticosteroid injection in addition to balloon dilatation has been studied, but it remains to be seen whether long-term prevention of re-stenosis occurs. Other endoscopic therapies and new developments are also discussed in this chapter.

Short chain fatty acids stimulate epithelial mucin 2 expression through differential effects on prostaglandin E(1) and E(2) production by intestinal myofibroblasts.

BACKGROUND: The mucus layer protects the gastrointestinal mucosa from mechanical, chemical, and microbial challenge. Mucin 2 (MUC-2) is the most prominent mucin secreted by intestinal epithelial cells. There is accumulating evidence that subepithelial myofibroblasts regulate intestinal epithelial cell function and are an important source of prostaglandins (PG). PG enhance mucin secretion and are key players in mucoprotection. The role of bacterial fermentation products in these processes deserves further attention. AIMS: We therefore determined whether the effect of short chain fatty acids (SCFA) on MUC-2 expression involves intermediate PG production. METHODS: Both mono- and cocultures of epithelial cells and myofibroblasts were used to study the effects of SCFA on MUC-2 expression and PG synthesis. Cell culture supernatants were used to determine the role of myofibroblast derived prostaglandins in increasing MUC-2 expression in epithelial cells. RESULTS: Prostaglandin E(1) (PGE(1)) was found to be far more potent than PGE(2) in stimulating MUC-2 expression. SCFA supported a mucoprotective PG profile, reflected by an increased PGE(1)/PGE(2) ratio in myofibroblast supernatants and increased MUC-2 expression in mono- and cocultures. Incubation with indomethacin revealed the latter to be mediated by PG. CONCLUSIONS: SCFA can differentially regulate PG production, thus stimulating MUC-2 expression in intestinal epithelial cells. This mechanism involving functional interaction between myofibroblasts and epithelial cells may play an important role in the mucoprotective effect of bacterial fermentation products.

Interleukin-6, C-reactive protein, and expression of human leukocyte antigen-DR on peripheral blood mononuclear cells in patients after laparoscopic vs. conventional bowel resection: a randomized study.

PURPOSE: The aim of the study was to investigate the effect of surgical trauma in terms of approach (laparoscopic vs. conventional surgery) and extent of bowel resection (ileocolic resection vs. colectomy) on interleukin-6 level, C-reactive protein level, and expression of human leukocyte antigen-DR on peripheral blood mononuclear cells. Second, the length of the incision was correlated with the inflammatory response. METHODS: Thirty-four patients were analyzed as part of a randomized trial comparing laparoscopically assisted vs. open bowel resection for Crohn's disease, ulcerative colitis, and familial adenomatous polyposis. C-reactive protein levels and expression of human leukocyte antigen-DR on peripheral blood mononuclear cells were measured preoperatively and one day after surgery. Interleukin-6 was measured preoperatively and on Days 1 and 7 postoperatively. RESULTS: Four of the 34 patients were excluded because of blood transfusion after surgery. One day postoperatively, the interleukin-6 level peaked significantly within the laparoscopic and conventional group. There was no significant difference between the conventional and laparoscopic groups at Day 1 postoperatively. At Day 7 postoperatively, interleukin-6 levels were similar in both groups and returned to baseline levels. There was a higher C-reactive protein level in the conventional group one day after surgery than in the laparoscopic group, although the difference was not significant. Preoperative and postoperative human leukocyte antigen-DR expression on monocytes and postoperative percentage of lymphocytes expressing human leukocyte antigen-DR did not differ between the conventional and laparoscopic groups. No differences in immune response with respect to the measured parameters were noticed in patients with a large or small bowel resection segment or in patients with a small (8 cm) incision. CONCLUSIONS: These data suggest that surgical trauma did not significantly affect the immune status of patients with respect to the measured parameters in terms of either the approach or the extent of bowel resection.

Infliximab therapy in Crohn's disease: safety issues.

Infliximab has become a valuable addition to the therapeutic arsenal for Crohn's disease. Although the rate of adverse events was relatively low in the premarketing trials, several investigators have recently reported experience in large groups of patients. This has shed more light on safety aspects of infliximab therapy, which should change the approach towards patients prior to infliximab infusion. This review discusses some immunological aspects that are relevant for infliximab therapy and provides guidelines for daily practice.

Regulation of the p21Ras-MAP kinase pathway by factor VIIa.

BACKGROUND: In recent years it has become clear that factor (F)VIIa is not a passive mediator involved in the linear transduction of the coagulation cascade, but actively engages target cells to induce signal transduction and that this signal transduction fulfills critical functions in angiogenesis, arteriosclerosis and inflammatory processes. OBJECTIVES: The details of coagulation factor-dependent signal transduction are among the least understood in biology and thus we set out to establish the molecular events responsible for MAP kinase activation induced by the interaction of FVIIa with its cellular binding partner tissue factor (TF). METHODS: Two different TF-expressing cell types, BHKTF and HaCaT cells, were assayed for p21Ras activation using a pull-down assay that is specific for activated Ras. This activation was visualized by means of Western blotting. In addition, the upstream pathways leading to FVIIa-induced Ras activation were characterized using phosphospecific antibodies and specific inhibitors. RESULTS: We observed that in both BHKTF and HaCaT cells FVIIa-induced MAP kinase activation correlates with p21Ras activation, and that this p21Ras activation is essential for FVIIa-induced MAP kinase activation. In BHKTF cells, early p21Ras activation was mediated by the activation of protein kinase C (PKC), whereas late p21Ras activation employed alternative mechanisms. In HaCaT cells, stimulation of the Src kinase family mediated FVIIa-dependent p21Ras activation. Finally, in both cell types, Raf activity was mandatory for MAP kinase activation. CONCLUSIONS: p21Ras activation is instrumental in FVIIa signal transduction and the FVIIa-dependent activation of p21Ras involves either PKC or Src-dependent mechanisms, depending on the cell type investigated.

Ano-genital granulomatosis: the counterpart of oro-facial granulomatosis.

Ano-genital granulomatosis encompasses the previously recognized clinical entities of vulvitis granulomatosa, posthitis granulomatosa, and ano-perineitis granulomatosa. We report three patients with ano-genital granulomatosis. The pathological features of the disease are lymphoedema and the presence of non-caseating giant cell granulomas. These granulomas are histologically indistinguishable from those found in both Crohn's disease and sarcoidosis, therefore, patients with ano-genital granulomatosis with accompanying gastro-intestinal or pulmonary symptoms should be investigated for the presence of Crohn's disease or sarcoidosis, respectively. The value of ano-genital granulomatosis as a unifying clinicopathologic concept is to provide a label for the affliction as well as to stimulate a careful search for possibly underlying systemic disorders, thus also permitting a more specific approach to therapy.

New biological therapies in inflammatory bowel disease.

Several biological therapies (monoclonal antibodies, designer molecules, recombinant cytokines) have been tested for clinical efficacy in inflammatory bowel disease, and some have been found to be effective. Anti-TNF-alpha (anti-tumour necrosis factor-alpha) antibody therapy is an important treatment modality in the treatment of active and fistulating Crohn's disease and should be considered in patients who fail standard medical therapies. Treatment with TNF-alpha-neutralizing antibodies is associated with immunosuppression that may lead to opportunistic infections and reactivation of tuberculosis, and patients should undergo Mantoux testing prior to treatment. Several other monoclonal antibodies, including anti-IL12 and anti-IFN-gamma, are currently in development for Crohn's disease. Other new approaches include ex vivo generation of regulatory T lymphocytes and antibodies that target and kill (subpopulations of) memory T lymphocytes.

Mitogen activated protein (MAP) kinase signal transduction pathways and novel anti-inflammatory targets.

Over the last decade important advances have been made in our understanding of the molecular events underlying cellular responses to extracellular signals. Increased understanding of signal transduction mechanisms and gene regulation involved in immune responses has created opportunities for the discovery of novel therapeutic compounds useful in treating inflammatory disorders. One of the best studied signalling routes is the mitogen activated protein (MAP) kinase signal transduction pathway which plays a crucial role in many aspects of immune mediated inflammatory responses. Here, our current understanding of the MAP kinase pathway is reviewed, as well as recent advances in the design of novel agents that are able to modulate the activity of these signalling cascades.

Taming the mucosal immune response in Crohn's disease.

New effective therapeutic strategies for inflammatory bowel disease are based on recent knowledge of the regulation of the immune response. Specific defects of innate immunity, such as the NOD2 mutation in a subset of patients with Crohn's disease, have been associated with inflammatory bowel disease and provide new therapeutic targets. The ultimate therapeutic goal is the complete restoration of the mucosal immune balance and healing of all intestinal lesions. This may require repair of the underlying genetic mutation, restoration of defects of apoptosis, or generation of regulatory T-lymphocytes.

Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract.

BACKGROUND: Sonic hedgehog (Shh) is an important endodermal morphogenetic signal during the development of the vertebrate gut. It controls gastrointestinal patterning in general, and gastric gland formation in particular. We have previously shown that Shh regulates gastric gland proliferation in the adult but detailed analysis of its expression along the adult gastrointestinal tract has never been undertaken. We therefore studied Shh expression along the normal human and rodent adult gastrointestinal tract as well as in intestinal metaplasia of the stomach, gastric and intestinal metaplasia of the oesophagus, and gastric heterotopia in Meckel's diverticulum. METHODS: The studies were performed with in situ hybridisation and by immunohistochemistry using an antibody that recognises the Shh precursor form. RESULTS: We found that in the normal gastrointestinal tract, high levels of Shh were expressed in the fundic glands of the stomach. Shh expression was also found in fundic gland metaplasia and heterotopia. However, Shh expression was lost in intestinal metaplasia of the stomach. CONCLUSION: We found a strong correlation between Shh expression and fundic gland differentiation. Our current study therefore provides evidence that in addition to its role in gastric epithelial development, Shh plays a unique role in gastric epithelial differentiation in adults.

A coculture model mimicking the intestinal mucosa reveals a regulatory role for myofibroblasts in immune-mediated barrier disruption.

The pathogenesis of Crohn's disease involves a mucosal inflammatory response affecting the barrier function of the gut. Myofibroblasts directly underlining the intestinal epithelium may have a regulatory role in immune-mediated barrier disruption. A coculture system of T84 epithelial and CCD-18Co myofibroblasts was established in order to mimic the in situ spatial interactions between these cell types and to evaluate their role in barrier: integrity. Lamina propria mononuclear cells (LPMC) were introduced in co- and monocultures. Effects of immune cells on barrier integrity was determined by measuring resistance and permeability for macromolecules. Introduction of LPMC in both culture systems caused a time-dependent decrease in barrier integrity. This was found to be less pronounced in cocultures indicating a regulatory role for mesenchymal cells. The effects were also found to depend on the route of LPMC stimulation. Additional analyses suggested that the regulatory role of myofibroblasts in barrier integrity involves production of growth factors.

Effects of anti-tumour necrosis factor-alpha therapy on the quality of life in Crohn's disease.

BACKGROUND: Infusion of anti-tumour necrosis factor-alpha appears to be highly effective in patients with Crohn's disease. AIM: To assess the effect of infliximab on the quality of life in patients with active or fistulizing disease, as measured by the inflammatory bowel disease questionnaire, and to examine the impact on its four dimensions. METHODS: An observational study was conducted in 65 patients. An infusion of 5 mg/kg infliximab was given at week 0 in patients with active disease and at week 0, 2 and 6 in fistulizing disease. Changes from baseline in the total and dimensional inflammatory bowel disease questionnaire scores were calculated and compared between the patient groups. Potential predictors of change in the quality of life were identified. RESULTS: In the active disease group, at week 4, the mean total and dimensional inflammatory bowel disease questionnaire scores improved compared to baseline (P < 0.001). In the fistulizing group, at week 6, all scores changed from baseline (P < 0.05). Improvement in the total inflammatory bowel disease questionnaire score correlated well with the improvement of the Crohn's disease activity index. Systemic and social scores improved more than bowel and emotional scores. Inflammatory Crohn's disease and a young age at diagnosis were predictors for a better response to infliximab therapy. CONCLUSIONS: Infliximab therapy improves all dimensions of the quality of life in patients with Crohn's disease.

Small therapeutic molecules for the treatment of inflammatory bowel disease.

New therapies for inflammatory bowel disease are needed, because standard therapies fail to induce remission in about 30% of patients, and because of the relative inefficacy of current maintenance therapies. This review summarises the current status of the development of small therapeutic molecules for inflammatory bowel disease.

Dichotomal role of inhibition of p38 MAPK with SB 203580 in experimental colitis.

BACKGROUND: Crohn's disease is characterised by a chronic relapsing inflammation of the bowel in which proinflammatory cytokines play an important perpetuating role. Mitogen activated protein kinase p38 (p38 MAPK) has been established as a major regulator of the inflammatory response, especially with regard to production of proinflammatory cytokines, but its role in inflammatory bowel disease is unexplored. In this paper we describe the effects of a specific p38 MAPK inhibitor, SB 203580, in trinitrobenzene sulphonic acid (TNBS) induced colitis in mice. RESULTS: SB 203580 had a dichotomal effect in TNBS mice. Weight loss of TNBS mice treated with SB 203580 was significantly worse and colon weight on sacrifice was significantly increased in MAPK inhibitor treated TNBS mice (229.2 mg and 289.1 mg, respectively). However, the total number of cells in the caudal lymph node decreased to 188.8 x 10(4) cells in SB 203580 treated TNBS mice compared with 334 x 10(4) cells in vehicle treated mice. CD3/CD28 double stimulated caudal lymph node cells of SB 203580 treated mice showed decreased interferon gamma production but increased tumour necrosis factor alpha production. The concentration of interleukin 12p70 in colon homogenates was significantly decreased in SB 203580 treated mice whereas concentrations of interleukin 12p40, tumour necrosis factor alpha, and interleukin 10 were similar in vehicle and SB 203580 treated TNBS mice. CONCLUSION: Our results reveal a dichotomy in p38 MAPK action during experimental colitis.