Elizabeth V. Fowler

Reduced alpha-defensin expression is associated with inflammation and not NOD2 mutation status in ileal Crohn’s disease

Background and aims: Reduced ileal Paneth cell α-defensin expression has been reported to be associated with Crohn’s disease, especially in patients carrying NOD2 mutations. The aim of this study was to independently assess whether NOD2, α-defensins and Crohn’s disease are linked. Methods: Using quantitative real-time polymerase chain reaction (RT-PCR), we measured the mRNA expression levels of key Paneth cell antimicrobial peptides (DEFA5, DEFA6, LYZ, PLA2G2A), inflammatory cytokines [interkelukin 6 (IL6) and IL8], and a marker of epithelial cell content, villin (VIL1) in 106 samples from both affected ileum (inflamed Crohn’s disease cases, n = 44) and unaffected ileum (non-inflamed; Crohn’s disease cases, n = 51 and controls, n = 11). Anti-human defensin 5 (HD-5) and haematoxylin/eosin immunohistochemical staining was performed on parallel sections from NOD2 wild-type and NOD2 mutant ileal Crohn’s disease tissue. Results: In Crohn’s disease patients, DEFA5 and DEFA6 mRNA expression levels were 1.9- and 2.2-fold lower, respectively, in histologically confirmed inflamed ileal mucosa after adjustment for confounders (DEFA5, p<0.001; DEFA6, p = 0.001). In contrast to previous studies, we found no significant association between α-defensin expression and NOD2 genotype. HD-5 protein data supports these RNA findings. The reduction in HD-5 protein expression appears due to surface epithelial cell loss and reduced Paneth cell numbers as a consequence of tissue damage. Conclusions: Reduction in α-defensin expression is independent of NOD2 status and is due to loss of surface epithelium as a consequence of inflammatory changes rather than being the inciting event prior to inflammation in ileal Crohn’s disease.

Prevalence of CARD15/NOD2 mutations in Caucasian healthy people

BACKGROUND:Crohns disease (CD) has been associated with CARD15/NOD2 mutations in Caucasians. The R702W, G908R, and 1007fs mutations represent 82% of the mutated chromosomes. The relative risk of developing CD in homozygous or compound heterozygous people has been estimated as between 10 and 40 times that of the general population. This high risk may support the opinion that CARD15/NOD2 variants are strong CD risk factors at the individual and population levels.SUBJECTS AND METHODS:The allele and genotype frequencies were calculated for the R702W, G908R, and 1007fs mutations in 3,575 Caucasian healthy controls recruited by 15 groups distributed on three continents. Geographic homogeneity was tested and the observed proportion of double mutants was compared with the expected value using χ2 tests.RESULTS:The allele frequencies of the R702W, G908R, and 1007fs mutations were 4.3% (3.6–4.9), 1.2% (0.8–1.6), and 2.3% (1.8–2.8), respectively, with large geographic fluctuations of the G908R, 1007fs, and wild-type alleles (P < 0.0001). At the population level, no simple relationship was observed between mutation frequencies and the disease incidences in the studied populations. At the individual level, no significant deficit of double-dose mutation carriers among healthy controls was found, providing strong evidence that the penetrances of the most at-risk genotypes are low.CONCLUSION:Altogether, these data confirm that CARD15/NOD2 acts in interaction with other unknown risk cofactors.

Clinically active Crohn's disease in the presence of a low C-reactive protein

Objective. Clinical interest in C-reactive protein (CRP) – a component of the innate immune system – has focused mainly on its worth as an indicator of disease activity. There has been a resurgence of interest in CRP in the Crohns disease (CD) literature because several trials of new treatments for active CD have been characterized by both a large proportion of patients with low CRP (<10 mg/l) at entry to the trials and by a negative therapeutic outcome. It is therefore of interest to study the clinical characteristics of patients who are thought to have at the same time both active CD and a low CRP. Material and methods. Patients were prospectively recruited as part of the Brisbane IBD clinical and research programme. Subjects were included in the low CRP group only if there were complete datasets for CRP on all occasions of active CD, and CRP was < 10 mg/l. Active disease was defined as CD activity index (CDAI) > 200. The low CRP group was compared with patients in the raised CRP group for a range of clinical variables as well as the major NOD2 variants. Results. There were data sets for 223 CD patients, with a mean disease duration of 12 years. Of these, 22 patients fulfilled the criteria for low CRP. The low CRP group (group 1) showed significant differences for disease site (p<0.01) and for BMI (p=0.006) compared to the raised CRP group (group 2). Specifically, group 1 had a predominance of pure ileal disease (95% versus 53%) and lack of pure colonic disease (0% versus 24%) compared to group 2, and their BMI was significantly lower (20.3 kg/m2 versus 25.0 kg/m2). Groups 1 and 2 did not differ with respect to Vienna behaviour at diagnosis, smoking, appendicectomy, extra-intestinal manifestations of CD, or NOD2 SNP variants. There was a trend for low CRP patients with previous ileal resection to evolve to a stricturing phenotype. Fat wrapping was noted in 11/13 (85%) of low CRP patients undergoing ileal resections. Conclusions. Patients with CD and a persistently low CRP in the face of active disease were characterized by an almost exclusive ileal disease distribution and a low BMI, compared to those with a raised CRP. These patients had a similar frequency and distribution of NOD2/CARD15 variants. Stricturing (v inflammatory or penetrating) behaviour may explain some low CRP. Despite the abnormally low BMI, fat wrapping was noted in the majority of low CRP patients undergoing ileal resection.

ATG16L1 T300A Shows Strong Associations With Disease Subgroups in a Large Australian IBD Population: Further Support for Significant Disease Heterogeneity

OBJECTIVES:Crohns disease (CD) and ulcerative colitis (UC) are the two most common forms of inflammatory bowel disease (IBD), representing a significant health-care burden. A variant in the autophagy gene ATG16L1 (T300A) has been newly identified as a CD susceptibility locus by genome-wide association. Our aim was to assess the contribution of T300A in determining disease susceptibility and phenotype in two independent Australian IBD cohorts and explore the relationship between T300A and known CD risk factors (NOD2[ nucleotide-binding oligomerization domain containing 2] status and smoking).METHODS:In total, 669 CD and 543 UC cases, and 1,244 controls (study 1), 154 CD cases and 420 controls (study 2), and 702 unaffected parents from both groups were genotyped. We conducted case–control and family association analyses, and investigated relationships between T300A and disease subgroups and between NOD2 status and cigarette smoking (CD only).RESULTS:The strong association between CD and T300A was confirmed (P < 0.001), with a two-fold increase in disease risk associated with the GG genotype (odds ratio [OR] 1.96, 95% confidence interval [CI] 1.49–2.58), while ileal CD risk was almost three-fold (OR 2.73, CI 1.87–4.0). ATG16L1 and NOD2 were found to contribute independently to CD risk. A greater than seven-fold increased CD risk was observed for current smokers with a GG genotype (vs nonsmoking AA genotype; P < 0.001, OR 7.65, CI 4.21–13.91). A significant inverse association was found between T300A and UC (P= 0.002). This was strongest for patients with extensive, severe disease.CONCLUSIONS:We confirm the strong association between T300A and CD, specifically ileal subphenotype, and also report the first strong association of this variant with UC.

KCNN4 gene variant is associated with ileal Crohn's Disease in the Australian and New Zealand population.

OBJECTIVES:Crohns disease (CD; MIM 266600) is one of the most common forms of inflammatory bowel disease (IBD), and represents a significant burden to health care in developed countries. Our aim was to determine whether a gene in the IBD linkage region on chromosome 19q13, with a role in Paneth cell secretion and T-cell activation, conferred genetic susceptibility to the development of CD.METHODS:In total, 792 CD cases and 1,244 controls of Australian origin (Caucasian) were genotyped for seven single-nucleotide polymorphisms (SNPs) in the gene encoding the intermediate conductance calcium-activated potassium channel protein (KCNN4) at 19q13.2. CD cases were phenotyped using the Montreal classification. The replication set comprised an additional 326 CD cases and 951 population-based Caucasian controls. Analysis of the KCNN4 mRNA transcript was carried out using quantitative reverse transcriptase-PCR.RESULTS:KCNN4 SNP rs2306801 was associated with CD (primary P=0.0008, odds ratio (OR) (95% confidence interval (CI)): 0.76 (0.65–0.89); replication P=0.01, OR (95% CI): 0.77 (0.61–0.97). Stratification by disease location identified the association between SNP rs2306801 and ileal CD (P=0.01). Non-inflamed ileal mucosa from CD patients carrying any of the common disease-predisposing NOD2 variants (R702W, G908R, 1007fs) had significantly reduced levels of KCNN4 mRNA expression (P=0.001). KCNN4 protein expression was detected in Paneth cells, and in T cells in inflamed lamina propria.CONCLUSIONS:Our data implicate the role of KCNN4 in ileal CD. The dual roles of KCNN4 in Paneth cell secretion and T-cell activation and also its nature as a potassium channel make it an important and practical therapeutic target.

Novel NOD2 haplotype strengthens the association between TLR4 Asp299gly and Crohn's disease in an Australian population

Background: The first major Crohns disease (CD) susceptibility gene, NOD2, implicates the innate intestinal immune system and other pattern recognition receptors in the pathogenesis of this chronic, debilitating disorder. These include the Toll‐like receptors, specifically TLR4 and TLR5. A variant in the TLR4 gene (A299G) has demonstrated variable association with CD. We aimed to investigate the relationship between TLR4 A299G and TLR5 N392ST, and an Australian inflammatory bowel disease cohort, and to explore the strength of association between TLR4 A299G and CD using global meta‐analysis. Methods: Cases (CD = 619, ulcerative colitis = 300) and controls (n = 360) were genotyped for TLR4 A299G, TLR5 N392ST, and the 4 major NOD2 mutations. Data were interrogated for case‐control analysis prior to and after stratification by NOD2 genotype. Genotype–phenotype relationships were also sought. Meta‐analysis was conducted via RevMan. Results: The TLR4 A299G variant allele showed a significant association with CD compared to controls (P = 0.04) and a novel NOD2 haplotype was identified which strengthened this (P = 0.003). Furthermore, we identified that TLR4 A299G was associated with CD limited to the colon (P = 0.02). In the presence of the novel NOD2 haplotype, TLR4 A299G was more strongly associated with colonic disease (P < 0.001) and nonstricturing disease (P = 0.009). A meta‐analysis of 11 CD cohorts identified a 1.5‐fold increase in risk for the variant TLR4 A299G allele (P < 0.00001). Conclusions: TLR 4 A299G appears to be a significant risk factor for CD, in particular colonic, nonstricturing disease. Furthermore, we identified a novel NOD2 haplotype that strengthens the relationship between TLR4 A299G and these phenotypes.

Smoking behaviour modifies IL23r-associated disease risk in patients with Crohn's disease

The etiology of Crohns disease (CD) implicates both genetic and environmental factors. Smoking behavior is one environmental risk factor to play a role in the development of CD. The study aimed to assess the contribution of the interleukin 23 receptor (IL23R) in determining disease susceptibility in two independent cohorts of CD, and to investigate the interactions between IL23R variants, smoking behavior, and CD‐associated genes, NOD2 and ATG16L1.

Tumor necrosis factor‐α haplotype is strongly associated with bone mineral density in patients with Crohn's disease

Background and Aim:  There is limited consensus on the major variables that determine bone integrity and bone loss in patients with Crohns disease. Twin and family studies in the general population indicate that up to 85% of variance in bone mineral density is inherited. The aim was to determine the prevalence of bone loss and both molecular and clinical risk factors for bone loss in a large Crohns disease population.

Altered Expression of Angiotensinogen and Mediators of Angiogenesis in Ileal Crohn's Disease.

BACKGROUND AND AIMS Angiotensin II (AII) is a powerful splanchnic vasoconstrictor with pro-inflammatory and pro-fibrotic properties. Angiotensin converting enzyme (ACE) inhibitors and AII Receptor Antagonists (ARBs) are therapeutic in animal models of colitis. The aim of this case-control study is to determine the expression of angiotensinogen and related genes in human ileal Crohns disease. METHODS Using quantitative real-time polymerase chain reaction (RT-PCR), we measured mRNA expression levels of angiotensinogen (AGT), hypoxia inducible factor (HIF)1α and melanoma cell adhesion molecule (MCAM; CD146) in 101 human samples (69 biopsy, 12 resection) from affected ileum (inflamed CD cases, n=36) and unaffected ileum (non-inflamed CD cases, n=45 and controls, n=20). Immunohistochemistry for angiotensinogen was also performed. The study was of case-control design in a tertiary care setting. RESULTS Ileal expression of AGT was lower in CD cases compared to controls (p<0.0001), in inflamed CD samples (p=0.017) and current smokers (p=0.02). HIF1α expression was lower in non-inflamed CD biopsy samples than controls (p=0.006). The presence of disease-associated NOD2 variants was associated with increased expression of markers of angiogenesis (HIF1α p=0.009; MCAM p=0.007) in inflamed CD samples. Angiotensinogen immunohistochemical staining supported the quantitative RT-PCR expression findings. CONCLUSIONS Angiotensinogen expression is down regulated in human ileal CD, particularly in the presence of inflammation and current cigarette smoking, implicating the mesenteric vasculature and mucosal hypoxia as co-factors in ileal CD pathogenesis. A novel reduction in HIF1α expression in non-inflamed ileal mucosa in CD patients was also demonstrated.

Is there a role for MICA in ulcerative colitis

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