Nathalie Vermeulen

Anti–α-enolase Antibodies in Patients with Inflammatory Bowel Disease

BACKGROUND Patients with inflammatory bowel disease (IBD) carry autoantibodies such as perinuclear antineutrophil cytoplasmic antibodies (pANCA). alpha-Enolase has been proposed as a target antigen in IBD. We evaluated the prevalence and diagnostic value of anti-alpha-enolase antibodies in IBD and related disorders. METHODS We used a classic proteomic approach with extracts from granulocytes and pANCA-positive ulcerative colitis (UC) sera to confirm alpha-enolase as a target antigen. By means of Western blot analysis, we screened a cohort of 525 subjects for the presence of anti-alpha-enolase antibodies. We performed GeneArray experiments on RNA extracted from colonic mucosal biopsies from 35 IBD and 6 control patients. RESULTS We detected anti-alpha-enolase antibodies 49.0% of patients with UC, 50.0% of patients with Crohns disease, 30.5% of patients with primary sclerosing cholangitis, 37.8% of patients with autoimmune hepatitis, 34.0% of patients with ANCA-positive vasculitis, 31.0% of non-IBD gastrointestinal controls, and 8.5% of healthy controls. Gene array experiments showed a significant upregulation of alpha-enolase mRNA in colonic mucosal biopsies from patients with IBD, but not from controls. There was no association between the presence of pANCA and anti-alpha-enolase antibodies. Preabsorption with alpha-enolase did not eliminate the pANCA pattern on indirect immunofluorescence. CONCLUSIONS Anti-alpha-enolase antibodies are present in a substantial proportion of patients with IBD, patients with various inflammatory/autoimmune disorders, and non-IBD gastrointestinal controls. Therefore, anti-alpha-enolase antibodies are of limited diagnostic value for the diagnosis of IBD.

Identification of a novel autoantigen in inflammatory bowel disease by protein microarray.

Background: Patients with inflammatory bowel disease (IBD) display immunoreactivity to self‐antigens and microbial antigens. We used a protein microarray approach to identify novel autoantigens in IBD. Methods: ProtoArray Human Protein Microarray v4.0 containing 8268 human proteins from Invitrogen (La Jolla, CA) was used. Results: Twenty‐five IBD patients and five healthy controls were screened for candidate autoantigens. For 256 antigens, IBD patients had a higher seroreactivity than controls. Twenty antigens were selected for further evaluation in a larger cohort (60 ulcerative colitis [UC] patients, 60 Crohns disease [CD] patients, 60 healthy controls, and 60 gastrointestinal‐diseased controls) by means of a customized protein microarray. Out of these 20 antigens, one antigen, family with sequence similarity 84 member A (FAM84A), was identified as a target antigen in IBD. Antibodies to FAM84A were significantly more prevalent in IBD patients (19%) than in gastrointestinal‐diseased controls (1.7%) (P = 0.0008) and healthy controls (5%) (P = 0.01). Anti‐FAM84A antibodies were found in 26.6% of UC patients and in 11.7% of CD patients. FAM84A was confirmed as target antigen in IBD by means of Western blotting in a large independent cohort (100 UC patients, 106 CD patients, 102 healthy controls, and 100 gastrointestinal‐diseased controls). Antibodies to FAM84A were significantly more prevalent in IBD patients (20%) than in gastrointestinal‐diseased controls (5%) (P = 0.0004) and healthy controls (0%) (P < 0.0001). Anti‐FAM84A antibodies were found in 18% of UC patients and in 22% of CD patients. Conclusions: We identified FAM84A as a novel autoantigen in IBD. (Inflamm Bowel Dis 2011;)

Auto)Antibodies in Inflammatory Bowel Diseases

Patients who have inflammatory bowel diseases (IBD) express strong antibody responses to a variety of epitopes. A number of (auto)antibodies have been described in patients who have Crohns disease or ulcerative colitis. These markers reflect a loss of tolerance toward bacterial and fungal flora and have been studied for their clinical value in IBD patients. However, currently, they have no place in the diagnostic work up. Their real promise may lie in their use as surrogate markers of complicated aggressive disease as shown in various retrospective studies, but prospective data are lacking.

Seroreactivity against glycolytic enzymes in inflammatory bowel disease

Background: Patients with inflammatory bowel disease (IBD) carry autoantibodies such as perinuclear antineutrophil cytoplasmic antibodies. The aim of the current study was to further characterize the immune reactivity in IBD. Methods: We used an immunoproteomic approach with extracts from granulocytes and serum from ulcerative colitis (UC) patients and controls to identify target antigens. By means of Western blot analysis, we screened 60 UC and 60 Crohns disease (CD) patients, 60 diseased, and 60 healthy controls for the antibodies. We performed gene array experiments on RNA extracted from colonic mucosal biopsies from 42 IBD patients and six controls. Results: We identified aldolase A, phosphoglycerate mutase, alpha‐enolase, triose‐phosphate isomerase, and malate dehydrogenase as target antigens in IBD. Seroreactivity to at least one of these five antigens was detected in 53.3% of UC patients, 38.3% of CD patients, and 8.3% of controls. Seroreactivity to at least two antigens was detected in 16.7% of UC patients, 11.7% of CD patients, and none of the controls. Gene array experiments showed a significant upregulation of aldolase A, phosphoglycerate mutase, alpha‐enolase, and pyruvate kinase mRNA in biopsies from IBD patients, but not controls. UC and CD patients also showed enhanced expression of hypoxia‐inducible factor‐1, a transcription factor that induces expression of glycolytic enzymes. Conclusions: IBD patients show strong seroreactivity toward enzymes involved in the glycolysis. IBD patients also have increased colonic mRNA expression of glycolytic enzymes, which is triggered by hypoxia through the transcription factor HIF‐1. Inflamm Bowel Dis 2011

Likelihood ratio for Crohn's disease as a function of Anti-Saccharomyces cerevisiae antibody concentration

To the Editor: Anti-Saccharomyces cerevisiae antibodies (ASCA) are found in patients with Crohn’s disease (CD).1 The sensitivity and specificity of ASCA for CD has been reported to range between 41% and 76% and between 88% and 97.5%, respectively.2–4 Studies that addressed the clinical value of ASCA used a single cutoff value. In the present report we illustrate how likelihood ratios for ASCA depend on the antibody level. Our calculations were based on a clinically well-defined group of CD patients (n 57), ulcerative colitis patients (n 57), diseased control patients (including patients with diarrheal illnesses) (n 56), and healthy controls (n 56). The characteristics of the patients and controls are described in detail in Ref. 4. ASCA was assayed using 2 assays in which simultaneous measurement of IgG and IgA antibodies was performed: Alphadia ASCA screen enzyme-linked immunosorbent assay (ELISA) (IgA-IgG) (cutoff: 25 units/mL) (Alphadia, Wavre, Belgium) and AESKULISA Crohn’s check (cutoff: 24 units/mL) (Aesku.Diagnostics, Wendelsheim, Germany). Table 1 summarizes the sensitivities and specificities of ASCA for several cutoffs. This illustrates that increasing the cutoff results in enhanced specificity and decreased sensitivity. Next, we calculated the likelihood for ASCA for several test result intervals for CD patients and controls. For CD patients the likelihoods for the Alphadia assay were 59.6%, 12.3%, and 28.1%, for 20 units/mL, between 20 and 25 units/mL, and 25 units/mL, respectively. For controls (UC patients, healthy, and diseased controls) the values were 98.8%, 0.6%, and 0.6%, respectively. For each test result interval, the likelihood ratio (i.e., the likelihood for CD patients divided by the likelihood for controls) was calculated. The likelihood ratios for the Alphadia ASCA were 0.6 (95% confidence interval [CI]: 0.5–0.7), 20.7 (95% CI: 2.6–165.0), and 47.4 (95% CI: 6.4–349.8) for 20 units/mL, between 20 and 25 units/mL, and 25 units, respectively.5,6 The results are shown in Figure 1A. A similar analysis was performed for the Aesku Diagnostics assay (Fig. 1B). The likelihood ratios were 0.4 (95% CI: 0.3–0.6), 3.8 (95% CI: 2.0–7.2), and 25.2 (95% CI: 6.0–105.7) for 24 units/mL, 24–96 units/mL, and 96 units/mL, respectively. These data illustrate that the likelihood ratios increase with increasing antibody levels. These data also illustrate that a negative test result with the Alphadia assay has almost no clinical utility and cannot be used to exclude the diagnosis of CD (likelihood ratio of 0.6). A value 20 units/mL with the Alphadia assay significantly affects the posttest probability (likelihood ratio 20). This is even more pronounced for values 25 units (likelihood ratio 47.5), which strongly suggests the presence of CD. For the Aesku Diagnostics assay, a negative test result modestly affects the posttest probability of CD (likelihood ratio 0.2). A value between 24 and 96 units/mL indicates only a small (but relevant) difference in pretest to posttest probability (likelihood ratio of 4.5). By contrast, a highly elevated ASCA value ( 94 units/mL) (likelihood ratio of 27.7) will significantly affect posttest probability and strongly suggests CD. For each test result interval of ASCA, the posttest probability for CD was calculated as a function of the pretest probability. The results are shown in Figure 1C,D. In conclusion, we illustrated how likelihood ratios and posttest probabilities depend on antibody level for ASCA. Clinical laboratories might consider providing likelihood ratios and probability graphs for test result intervals. Clinicians might find such information clinically helpful.

Stability Of Gut Microbiota Over Time In Crohn's Disease Patients Compared To Healthy Relatives

well plates. BMDC were treated with media alone, E. coli LPS (1 μg/mL) or murine CpG (20 μM) for 24 hours, after which culture supernatant was harvested for cytokine ELISA. BMDC were stained for cell surface markers of activation and analyzed by flow cytometry. Results: LPS treated FVB.mdr1a-/BMDC cultures had greater amounts of IL-12 (679±149 vs. 488±73, pg/mL) but similar levels of TNF-α compared to FVB.WT. FVB.mdr1a-/BMDC cultured with CpG also produced more IL-12 (98±28 pg/mL vs. non-detectable) and TNFα (418±91 vs. 207±40, pg/mL). The percentage and absolute number of CD11c+CD11b+ cells were greater in CpG treated FVB.mdr1a-/BMDC. The levels of BMDC activation as determined as being CD11c+ CD80+ and CD11c+ CD80+, were greater in the LPS and CpG treated FVB.mdra1-/BMDC. Conclusions: TLR4 and TLR9 ligand treatment results in a greater activation of BMDC from FVB.mdra1-/-. Since these TLRs are present in the intracellular compartment the absence of p-glycoprotein (an ATP-dependent pump) to expel LPS or CpG from intestinal dendritic cells could contribute to the mechanism of colitis in FVB.mdra1-/mice.