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1.  Infliximab Exerts No Direct Hepatotoxic Effect on HepG2 Cells In Vitro 
Digestive Diseases and Sciences  2012;57(6):1604-1608.
Infliximab-induced hepatotoxicity is reported in several case studies involving patients with inflammatory bowel disease (IBD) and a direct hepatotoxic effect has been proposed.
The aim of this study was to determine the direct in vitro toxicity of infliximab. As a proof of principle the in vitro toxicity of thiopurines and methotrexate was also determined.
Cell survival curves and the half maximal inhibitory concentrations (IC50) were obtained after 24, 48 and 72 h of incubation in HepG2 cells with the IBD drugs azathioprine, 6-mercaptopurine, 6-thioguanine, methotrexate or infliximab by using the WST-1 cytotoxicity assay.
No in vitro hepatotoxicity in HepG2 cells was seen with infliximab, while concentration-dependent cytotoxicity was observed when HepG2 cells were incubated with increasing concentrations of azathioprine, 6-mercaptopurine and 6-thioguanine.
Infliximab alone or given in combination with azathioprine showed no direct hepatotoxic effect in vitro, indicating that the postulated direct hepatotoxicity of infliximab is unlikely.
PMCID: PMC3358583  PMID: 22535279
Inflammatory bowel disease; Hepatotoxicity; HepG2 cells; Infliximab; Methotrexate; Azathioprine; 6-mercaptopurine; 6-thioguanine
2.  Differential Toll-Like Receptor Recognition and Induction of Cytokine Profile by Bifidobacterium breve and Lactobacillus Strains of Probiotics ▿ 
The use of probiotics as a food supplement has gained tremendous interest in the last few years as beneficial effects were reported in gut homeostasis and nutrient absorption but also in immunocompromised patients, supporting protection from colonization or infection with pathogenic bacteria or fungi. As a treatment approach for inflammatory bowel diseases, a suitable probiotic strain would ideally be one with a low immunogenic potential. Insight into the immunogenicities and types of T-cell responses induced by potentially probiotic strains allows a more rational selection of a particular strain. In the present study, the bacterial strains Bifidobacterium breve (NumRes 204), Lactobacillus rhamnosus (NumRes1), and Lactobacillus casei (DN-114 001) were compared concerning their capacity to induce inflammatory responses in terms of cytokine production by human and mouse primary immune cells. It was demonstrated that the B. breve strain induced lower levels of the proinflammatory cytokine gamma interferon (IFN-γ) than the tested L. rhamnosus and L. casei strains. Both B. breve and lactobacilli induced cytokines in a Toll-like receptor 9 (TLR9)-dependent manner, while the lower inflammatory profile of B. breve was due to inhibitory effects of TLR2. No role for TLR4, NOD2, and C-type lectin receptors was apparent. In conclusion, TLR signaling is involved in the differentiation of inflammatory responses between probiotic strains used as food supplements.
PMCID: PMC3122558  PMID: 21288993
3.  Farnesoid X Receptor (FXR) Activation and FXR Genetic Variation in Inflammatory Bowel Disease 
PLoS ONE  2011;6(8):e23745.
We previously showed that activation of the bile salt nuclear receptor Farnesoid X Receptor (FXR) protects against intestinal inflammation in mice. Reciprocally, these inflammatory mediators may decrease FXR activation. We investigated whether FXR activation is repressed in the ileum and colon of inflammatory bowel disease (IBD) patients in remission. Additionally, we evaluated whether genetic variation in FXR is associated with IBD.
mRNA expression of FXR and FXR target gene SHP was determined in ileal and colonic biopsies of patients with Crohn's colitis (n = 15) and ulcerative colitis (UC; n = 12), all in clinical remission, and healthy controls (n = 17). Seven common tagging SNPs and two functional SNPs in FXR were genotyped in 2355 Dutch IBD patients (1162 Crohn's disease (CD) and 1193 UC) and in 853 healthy controls.
mRNA expression of SHP in the ileum is reduced in patients with Crohn's colitis but not in patients with UC compared to controls. mRNA expression of villus marker Villin was correlated with FXR and SHP in healthy controls, a correlation that was weaker in UC patients and absent in CD patients. None of the SNPs was associated with IBD, UC or CD, nor with clinical subgroups of CD.
FXR activation in the ileum is decreased in patients with Crohn's colitis. This may be secondary to altered enterohepatic circulation of bile salts or transrepression by inflammatory signals but does not seem to be caused by the studied SNPs in FXR. Increasing FXR activity by synthetic FXR agonists may have benefit in CD patients.
PMCID: PMC3161760  PMID: 21887309
4.  Effect of childbirth on the course of Crohn's disease; results from a retrospective cohort study in the Netherlands 
BMC Gastroenterology  2011;11:6.
Pregnant women with Crohn's disease needs proper counselling about the effect of pregnancy and childbirth on their disease. However, Literature about the effect of childbirth on Crohn's disease is limited. This study examined the effect of childbirth on the course of Crohn's disease and especially perianal Crohn's disease.
This is a retrospective cohort study which was performed in a tertiary level referral hospital in the Netherlands. From the IBD database, female patients aged 18-80 years in 2004 were selected. Data analysis took place in the years 2005 and 2006. Eventually, 114 women with at least one pregnancy after the diagnosis of Crohn's disease were eligible for the study. Differences between groups were analyzed using Wilcoxon Mann Whitney tests and Chi-square analysis with 2 × 2 or 2 × 3 contingency tables. Two-tailed values were used and p values < 0.05 were considered statistically significant.
21/114 women (18%) had active luminal disease prior to pregnancy, with significantly more pregnancy related complications compared to women with inactive luminal disease (Odds ratio 2.8; 95% CI 1.0 - 7.4). Caesarean section rate was relatively high (37/114, 32%), especially in patients with perianal disease prior to pregnancy compared to women without perianal disease (Odds ratio 4.6; 95% CI 1.8 - 11.4). Disease progression after childbirth was more frequent in patients with active luminal disease prior to pregnancy compared to inactive luminal disease (Odds ratio 9.7; 95% CI 2.1 - 44.3). Progression of perianal disease seems less frequent after vaginal delivery compared with caesarean section, in both women with prior perianal disease (18% vs. 31%, NS) and without prior perianal disease (5% vs 14%, NS). There were no more fistula-related complications after childbirth in women with an episiotomy or second degree tear.
A relatively high rate of caesarean sections was observed in women with Crohn's disease, especially in women with perianal disease prior to pregnancy. A protective effect of caesarean section on progression of perianal disease was not observed. However, this must be interpreted carefully due to confounder effect by indication for caesarean section.
PMCID: PMC3042417  PMID: 21269464
5.  The Functional −765G→C Polymorphism of the COX-2 Gene May Reduce the Risk of Developing Crohn's Disease 
PLoS ONE  2010;5(11):e15011.
Cyclooxygenase-2 (COX-2) is a key enzyme involved in the conversion of arachidonic acid into prostaglandins. COX-2 is mainly induced at sites of inflammation in response to proinflammatory cytokines such as interleukin-1α/β, interferon-γ and tumor necrosis factor-α produced by inflammatory cells.
The aim of this study was to investigate the possible modulating effect of the functional COX-2 polymorphisms −1195 A→G and −765G→C on the risk for development of inflammatory bowel disease (IBD) in a Dutch population.
Genomic DNA of 525 patients with Crohn's disease (CD), 211 patients with ulcerative colitis (UC) and 973 healthy controls was genotyped for the −1195 A→G (rs689466) and −765G→C (rs20417) polymorphisms. Distribution of genotypes in patients and controls were compared and genotype-phenotype interactions were investigated.
The genotype distribution of the −1195A→G polymorphism was not different between the patients with CD or UC and the control group. The −765GG genotype was more prevalent in CD patients compared to controls with an OR of 1.33 (95%CI 1.04–1.69, p<0.05). The −765GC and −765CC genotype carriers showed a tendency to be less frequent in patients with CD compared to controls, with ORs of 0.78 (95%CI: 0.61–1.00) and 0.49 (95%CI 0.22–1.08), respectively. Combining homozygous and heterozygous patients with the −765C allele showed a reduced risk for developing CD, with an OR of 0.75 (95%CI: 0.59–0.96). In the context of this, the G−1195G−765/A−1195C−765 diplotype was significantly less common in patients with CD compared to controls, with an OR of 0.62 (95%CI: 0.39–0.98). For UC however, such an effect was not observed. No correlation was found between COX-2 diplotypes and clinical characteristics of IBD.
The −765G→C polymorphism was associated with a reduced risk for developing Crohn's disease in a Dutch population.
PMCID: PMC2991351  PMID: 21124790
6.  Genetic Variants in the Region Harbouring IL2/IL21 Associated to Ulcerative Colitis 
Gut  2009;58(6):799-804.
Genetic susceptibility is known to play a large part in the predisposition to the inflammatory bowel diseases (IBD) known as Crohn’s disease (CD) and ulcerative colitis (UC). The IL2/IL21 locus on 4q27 is known to be a common risk locus for inflammatory disease (shown in celiac disease, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus and psoriasis), while the roles that IL2 and IL21 play in the immune response also make them attractive candidates for inflammatory bowel disease. Our objective was to test for association between the IL2/IL21 locus and the inflammatory bowel diseases.
The four single nucleotide polymorphisms (SNPs) in the IL2/IL21 locus most associated to celiac disease were genotyped in 1590 IBD cases and 929 controls from the Netherlands, and then replicated in a North American cohort (2387 cases and 1266 controls) and an Italian cohort (805 cases and 421 controls), yielding a total of 4782 cases (3194 UC, 1588 CD) and 2616 controls. Allelic association testing and a pooled analysis using a Cochran-Mantel-Haenszel test were performed.
All four SNPs were strongly associated with UC in all three cohorts and reached genome-wide significance in the pooled analysis (rs13151961 p= 1.35×10−10, rs13119723 p= 8.60×10−8, rs6840978 p= 3.07×10−8, rs6822844 p= 2.77×10−9). We also found a moderate association with CD in the pooled analysis (p value range 0.0016–9.86×10−5).
We found a strong association for the IL2/IL21 locus with UC, which also confirms it as a general susceptibility locus for inflammatory disease.
PMCID: PMC2757103  PMID: 19201773
Inflammatory bowel disease; ulcerative colitis; Crohn’s disease; IL2; IL21
7.  Genetic Association Analysis of the Functional c.714T>G Polymorphism and Mucosal Expression of Dectin-1 in Inflammatory Bowel Disease 
PLoS ONE  2009;4(11):e7818.
Dectin-1 is a pattern recognition receptor (PRR) expressed by myeloid cells that specifically recognizes β-1,3 glucan, a polysaccharide and major component of the fungal cell wall. Upon activation, dectin-1 signaling converges, similar to NOD2, on the adaptor molecule CARD9 which is associated with inflammatory bowel disease (IBD). An early stop codon polymorphism (c.714T>G) in DECTIN-1 results in a loss-of-function (p.Y238X) and impaired cytokine responses, including TNF-α, interleukin (IL)-1β and IL-17 upon in vitro stimulation with Candida albicans or β-glucan. The aim of the present study was to test the hypothesis that the DECTIN-1 c.714T>G (p.Y238X) polymorphism is associated with lower disease susceptibility or severity in IBD and to investigate the level of dectin-1 expression in inflamed and non-inflamed colon tissue of IBD patients.
Paraffin embedded tissue samples from non-inflamed and inflamed colon of IBD patients and from diverticulitis patients were immunohistochemically stained for dectin-1 and related to CD68 macrophage staining. Genomic DNA of IBD patients (778 patients with Crohn's disease and 759 patients with ulcerative colitis) and healthy controls (n = 772) was genotyped for the c.714T>G polymorphism and genotype-phenotype interactions were investigated.
Principal Findings
Increased expression of dectin-1 was observed in actively inflamed colon tissue, as compared to non-inflamed tissue of the same patients. Also an increase in dectin-1 expression was apparent in diverticulitis tissue. No statistically significant difference in DECTIN-1 c.714T>G allele frequencies was observed between IBD patients and healthy controls. Furthermore, no differences in clinical characteristics could be observed related to DECTIN-1 genotype, neither alone, nor stratified for NOD2 genotype.
Our data demonstrate that dectin-1 expression is elevated on macrophages, neutrophils, and other immune cells involved in the inflammatory reaction in IBD. The DECTIN-1 c.714T>G polymorphism however, is not a major susceptibility factor for developing IBD.
PMCID: PMC2771910  PMID: 19915667
8.  Laparoscopic ileocolic resection versus infliximab treatment of distal ileitis in Crohn's disease: a randomized multicenter trial (LIR!C-trial) 
BMC Surgery  2008;8:15.
With the availability of infliximab, nowadays recurrent Crohn's disease, defined as disease refractory to immunomodulatory agents that has been treated with steroids, is generally treated with infliximab. Infliximab is an effective but expensive treatment and once started it is unclear when therapy can be discontinued. Surgical resection has been the golden standard in recurrent Crohn's disease. Laparoscopic ileocolic resection proved to be safe and is characterized by a quick symptom reduction.
The objective of this study is to compare infliximab treatment with laparoscopic ileocolic resection in patients with recurrent Crohn's disease of the distal ileum with respect to quality of life and costs.
The study is designed as a multicenter randomized clinical trial including patients with Crohn's disease located in the terminal ileum that require infliximab treatment following recent consensus statements on inflammatory bowel disease treatment: moderate to severe disease activity in patients that fail to respond to steroid therapy or immunomodulatory therapy. Patients will be randomized to receive either infliximab or undergo a laparoscopic ileocolic resection. Primary outcomes are quality of life and costs. Secondary outcomes are hospital stay, early and late morbidity, sick leave and surgical recurrence. In order to detect an effect size of 0.5 on the Inflammatory Bowel Disease Questionnaire at a 5% two sided significance level with a power of 80%, a sample size of 65 patients per treatment group can be calculated. An economic evaluation will be performed by assessing the marginal direct medical, non-medical and time costs and the costs per Quality Adjusted Life Year (QALY) will be calculated. For both treatment strategies a cost-utility ratio will be calculated. Patients will be included from December 2007.
The LIR!C-trial is a randomized multicenter trial that will provide evidence whether infliximab treatment or surgery is the best treatment for recurrent distal ileitis in Crohn's disease.
Trial registration
Nederlands Trial Register NTR1150
PMCID: PMC2533646  PMID: 18721465
9.  NOD2 and Toll-Like Receptors Are Nonredundant Recognition Systems of Mycobacterium tuberculosis 
PLoS Pathogens  2005;1(3):e34.
Infection with Mycobacterium tuberculosis is one of the leading causes of death worldwide. Recognition of M. tuberculosis by pattern recognition receptors is crucial for activation of both innate and adaptive immune responses. In the present study, we demonstrate that nucleotide-binding oligomerization domain 2 (NOD2) and Toll-like receptors (TLRs) are two nonredundant recognition mechanisms of M. tuberculosis. CHO cell lines transfected with human TLR2 or TLR4 were responsive to M. tuberculosis. TLR2 knock-out mice displayed more than 50% defective cytokine production after stimulation with mycobacteria, whereas TLR4-defective mice also released 30% less cytokines compared to controls. Similarly, HEK293T cells transfected with NOD2 responded to stimulation with M. tuberculosis. The important role of NOD2 for the recognition of M. tuberculosis was demonstrated in mononuclear cells of individuals homozygous for the 3020insC NOD2 mutation, who showed an 80% defective cytokine response after stimulation with M. tuberculosis. Finally, the mycobacterial TLR2 ligand 19-kDa lipoprotein and the NOD2 ligand muramyl dipeptide synergized for the induction of cytokines, and this synergism was lost in cells defective in either TLR2 or NOD2. Together, these results demonstrate that NOD2 and TLR pathways are nonredundant recognition mechanisms of M. tuberculosis that synergize for the induction of proinflammatory cytokines.
Tuberculosis is one of the most prevalent infections worldwide, with 2 billion people believed to be infected, and 2 million deaths each year. In addition to representing a major health care problem in developing countries, concern is also growing about the increased incidence of tuberculosis in developed countries, especially in immunocompromised patients such as those with AIDS, transplantation, and immunosuppressive therapy. The present study describes the pathways that enable leukocytes to recognize M. tuberculosis, and demonstrates for the first time that NOD2, member of a new class of intracellular receptors, is an independent recognition mechanism for mycobacteria. NOD2 acts together with the earlier-described Toll-like receptors for the activation of host defenses during the encounter of leukocytes with M. tuberculosis. Understanding the mechanisms through which the cells of the immune system recognize M. tuberculosis can be an important step in designing new therapeutic approaches, as well as improving the limited success of current vaccination strategies.
PMCID: PMC1291354  PMID: 16322770
10.  On tolerability and safety of a maintenance treatment with 6-thioguanine in azathioprine or 6-mercaptopurine intolerant IBD patients 
AIM: To determine the tolerability and safety profile of a low-dose maintenance therapy with 6-TG in azathioprine (AZA) or 6-mercaptopurine (6-MP) intolerant inflammatory bowel disease (IBD) patients over a treatment period of at least 1 year.
METHODS: Database analysis.
RESULTS: Twenty out of ninety-five (21%) patients discontinued 6-TG (mean dose 24.6 mg; mean 6-TGN level 540 pmol/8×108 RBC) within 1 year. Reasons for discontinuation were GI complaints (31%), malaise (15%) and hepatotoxicity (15%). Hematological events occurred in three patients, one discontinued treatment. In the 6-TG-tolerant group, 9% (7/75) could be classified as hepatotoxicity. An abdominal ultrasound was performed in 54% of patients, one patient had splenomegaly.
CONCLUSION: The majority of AZA or 6-MP-intolerant IBD patients (79%) is able to tolerate maintenance treatment with 6-TG (dosages between 0.3 and 0.4 mg/kg per d). 6-TG may still be considered as an escape maintenance immunosuppressant in this difficult to treat group of patients, taking into account potential toxicity and efficacy of other alternatives. The recently reported hepatotoxicity is worrisome and 6-TG should therefore be administered only in prospective trials.
PMCID: PMC4320368  PMID: 16222751
6-Thioguanine; Azathioprine; 6-Mercaptopurine; Crohn’s disease; Ulcerative colitis; Side effects; Tolerance; Safety

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