Evaluation of biomarkers for ulcerative colitis comparing two sampling methods: fecal markers reflect colorectal inflammation both macroscopically and on a cellular level.

OBJECTIVE: Simple, objective and inexpensive tools for the assessment of mucosal inflammation in ulcerative colitis (UC) are highly desirable. The aim of this study was to evaluate a broad spectrum of activity markers comparing two sampling methods: fecal samples and the mucosal patch technique. METHODS: Twenty patients with active UC and 14 healthy controls were characterized by means of clinical indices and endoscopy together with histology and immunohistochemistry on colorectal sections. Neutrophil myeloperoxidase (MPO), calprotectin, eosinophil cationic protein (ECP), eosinophil protein X (EPX/EDN) and IL-1ß were analyzed in fecal samples and rectal fluid collected by the patch technique. Nitric oxide (NO) was analyzed in rectal gas samples. Expression of activity markers on colorectal neutrophils and eosinophils were analyzed by flow cytometry. RESULTS: All fecal and patch markers were increased in UC patients compared with healthy controls. Fecal markers and the level of neutrophil activation correlated to disease activity, whereas patch markers did not. The best markers in terms of discriminative power were fecal MPO and IL-1ß. Fecal marker levels were related to sigmoidal histology scores and to neutrophil number and activation. Patch markers were related to rectal inflammation only. CONCLUSIONS: The levels of inflammation markers in feces and patch fluid distinctly reflected active inflammation in UC. The degree of disease activity was however best assessed by fecal markers, particularly MPO and IL-1ß. Fecal markers reflect colorectal inflammation both macroscopically and on a cellular level, and may be useful for the evaluation of subclinical inflammation. The applicability of patch markers is restricted to rectal inflammation.

Fecal eosinophil cationic protein as a marker of active disease and treatment outcome in collagenous colitis: a pilot study.

BACKGROUND AND AIMS: Fecal calprotectin (FC) is used as a marker for intestinal inflammation in inflammatory bowel disease (IBD) but there is no reliable marker for collagenous colitis (CC). We have previously demonstrated that the mucosal inflammation in CC is characterized by eosinophil activation, which is restored during budesonide treatment, but there is no enhanced neutrophil activity. The aim of this study was to evaluate the use of fecal eosinophil cationic protein (F-ECP) and eosinophil protein X (F-EPX) compared with the neutrophil-derived myeloperoxidase (F-MPO) and FC in patients treated for active CC. METHODS: Patients with active CC (n = 12) were studied before and after 3, 7, 28 and 56 days of budesonide treatment. Clinical symptoms and stool frequency were recorded, fecal samples were collected, and F-ECP, F-EPX, F-MPO and FC were measured at each occasion. RESULTS: All but one patient achieved remission. On inclusion 92%, 67%, 67% and 75% of the patients had elevated F-ECP, F-EPX, F-MPO and FC levels, respectively. All markers decreased during the treatment, particularly F-ECP and F-EPX, which decreased after only 3 days. At the end of the study 100%, 92%, 83% and 75% of the patients had normal F-ECP, F-EPX, F-MPO and FC values, respectively. CONCLUSION: F-ECP demonstrated the best discriminating capacity in detecting active CC. A normalized F-ECP and F-EPX may further be studied as a marker for successful treatment. During budesonide treatment there is a rapid fall in F-ECP and F-EPX, accompanied by clinical improvement, indicating an essential role for the eosinophil participating in the pathophysiology of CC.

Fecal markers of inflammation used as surrogate markers for treatment outcome in relapsing inflammatory bowel disease.

AIM: To evaluate fecal calprotectin (FC) as a surrogate marker of treatment outcome of relapse of inflammatory bowel disease (IBD) and, to compare FC with fecal myeloperoxidase (MPO) and fecal eosinophil protein X (EPX). METHODS: Thirty eight patients with IBD, comprising of 27 with ulcerative colitis (UC) and 11 with Crohn's disease (CD) were investigated before treatment (inclusion), and after 4 and 8 wk of treatment. Treatment outcomes were evaluated by clinical features of disease activity and endoscopy in UC patients, and disease activity in CD patients. In addition, fecal samples were analyzed for FC by enzyme-linked immunosorbent assay (ELISA), and for MPO and EPX with radioimmunoassay (RIA). RESULTS: At inclusion 37 of 38 (97%) patients had elevated FC levels (> 94.7 microg/g). At the end of the study, 31 of 38 (82%) patients fulfilled predefined criteria of a complete response [UC 21/27 (78%); CD 10/11 (91%)]. Overall, a normalised FC level at the end of the study predicted a complete response in 100% patients, whereas elevated FC level predicted incomplete response in 30%. Normalised MPO or EPX levels predicted a complete response in 100% and 90% of the patients, respectively. However, elevated MPO or EPX levels predicted incomplete response in 23% and 22%, respectively. CONCLUSION: A normalised FC level has the potential to be used as a surrogate marker for successful treatment outcome in IBD patients. However, patients with persistent elevation of FC levels need further evaluation. FC and MPO provide superior discrimination than EPX in IBD treatment outcome.

Increased fecal levels of chromogranin A, chromogranin B, and secretoneurin in collagenous colitis.

Interactions between the enteric nervous system and the immune system are suggested to play an important role in the pathophysiology of inflammatory bowel disease (IBD). This study aims to determine if chromogranin A (CgA), chromogranin B (CgB), and secretoneurin (SN) are detectable in feces (F) from patients with collagenous colitis (CC) and to compare the levels found in patients with ulcerative colitis (UC) and Crohn's disease (CD) before and during treatment. Patients with CC (n = 12) were studied before and after 3, 7, 28, and 56 days of treatment. Patients with IBD (UC, n = 21; CD, n = 11) were studied before and after 28 and 56 days of treatment. Clinical data were recorded, and fecal samples were collected at each occasion. F-CgA, F-CgB, and F-SN were measured by RIA. Eleven patients with CC, 21 with UC, and 10 with CD achieved remission. On inclusion, CC patients had higher levels of F-CgA, F-CgB, and F-SN than patients with IBD and controls. Patients with IBD expressed markedly lower levels of F-SN than controls. During treatment, F-SN in CC patients decreased to control levels but remained low in IBD patients. No change was found in F-CgA or F-CgB in any of the groups. In conclusion, CgA, CgB, and SN are detectable in feces, and CC patients express higher values than patients with IBD and controls. During treatment, F-SN decreased to control levels in CC. These findings suggest that the enteric nervous system is clearly involved in the pathophysiology of CC.

An extragranular compartment of blood eosinophils contains eosinophil protein X/eosinophil-derived neurotoxin (EPX/EDN).

Serum and plasma profiles of eosinophil protein X (EPX/EDN) and those of other eosinophil proteins differ in various conditions, suggesting a different mobilisation from storage granules. This work studied the subcellular localisation of EPX/EDN in non-primed and in vivo primed blood eosinophils from healthy and allergic subjects, during and out of the pollen season. Primed eosinophils contain easily mobilisable secretory proteins. By fractionation on sucrose density gradients, EPX/EDN localised in the specific granules as well as in a cytoplasmic extra-granular compartment of low equilibrium density that partially overlapped with vesicular structures, cytosolic proteins and plasma membranes. This compartment was clearly separate from the low density peak of ECP that increases during the pollen season. There were no significant differences in the amounts of EPX/EDN present in the low density peak of healthy and allergic subjects. Immuno-gold labelling electron microscopy showed EPX/EDN in specific granules, cytoplasm and associated to plasma membranes. In conclusion, substantial amounts of EPX/EDN localise in an extra-granular, low equilibrium density compartment of human eosinophils.

Subclinical inflammation with increased neutrophil activity in healthy twin siblings reflect environmental influence in the pathogenesis of inflammatory bowel disease.

BACKGROUND: The mechanisms behind increased fecal calprotectin (FC) in healthy relatives of patients with inflammatory bowel disease (IBD) are unknown. Our aims were to explore if there is a subclinical inflammation with increased neutrophil activity in healthy twin siblings in discordant twin pairs with IBD and to assess the influence of genetics in this context. METHODS: Nuclear factor kappa B (NF-κB) and neutrophil activity, based on myeloperoxidase (MPO) and FC, were analyzed in healthy twin siblings in discordant twin pairs with IBD and compared with healthy controls. NF-κB and MPO were assessed by immunohistochemistry and FC by enzyme-linked immunosorbent assay. RESULTS: In total, 33 of 34 healthy twin siblings were histologically normal. Increased NF-κB was more often observed in healthy twin siblings in discordant twin pairs with Crohn's disease (13/18 [73%]) and with ulcerative colitis (12/16 [75%]) than in healthy controls (8/45 [18%]). MPO was more often increased in healthy twin siblings in discordant pairs with Crohn's disease (12/18 [67%]) than in healthy controls (11/45 [24%]) and FC more often in healthy twin siblings in discordant pairs with ulcerative colitis (14/21 [67%]) than in healthy controls (6/31 [19%]). Interestingly, the observed differences remained when healthy monozygotic and dizygotic twin siblings were analyzed separately. CONCLUSIONS: We observed increased NF-κB, MPO, and FC in healthy twins in both monozygotic and dizygotic discordant pairs with IBD. These novel findings speak for an ongoing subclinical inflammation with increased neutrophil activity in healthy first-degree relatives.

A new method for the quantification of neutrophil and eosinophil cationic proteins in feces: establishment of normal levels and clinical application in patients with inflammatory bowel disease.

OBJECTIVES: The aims of this study were 1) to develop a valid method for the measurement of the eosinophil proteins eosinophil cationic protein (ECP) and eosinophil protein X (EPX) and neutrophil proteins myeloperoxidase and human neutrophil lipocalin (HNL) in feces and 2) to investigate their potential role as disease activity markers in inflammatory bowel disease (IBD). METHODS: Feces samples were obtained from 44 apparently healthy individuals (HIs), 18 patients with IBD (11 with ulcerative colitis [UC] and seven with Crohn's disease [CD]), and three with collagen colitis. The granulocyte markers were measured using immunoassays in supernatants from processed feces. RESULTS: ECP, myeloperoxidase, and, to a lesser degree, EPX and HNL were bound to the solid part of feces. However, feces homogenized in an extraction buffer containing the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide allowed an efficient recovery of the proteins (i.e., up to 100-fold increased levels compared to homogenization in saline). All four proteins were stable for at least 7 days at +6 degrees C and at least 3 days at +22 degrees C. The normal fecal geometric mean (95th percentile) levels of ECP, EPX, myeloperoxidase, and HNL were estimated to be, respectively, 1.69 microg/g (6.41), 0.57 microg/g (1.72), 3.54 microg/g (8.77), and 1.97 microg/g (4.91). Markedly increased feces levels of all markers (p < 0.0002), compared to HIs and CD patients, were observed in UC. However, the marker levels in CD patients were significantly increased relative to HIs (p < 0.05 to p < 0.0002). Increased levels of HNL and myeloperoxidase were also observed in the three collagen colitis patients. The discriminative capability between UC patients and HIs was somewhat superior for EPX and myeloperoxidase. CONCLUSIONS: The method described here takes into account the molecular properties of the granule proteins and the heterogeneity in feces consistency, which is a prerequisite for a valid and reproducible measurement of cationic granule proteins. We suggest that EPX and myeloperoxidase, when applied in IBD, are the best eosinophil and neutrophil markers for studying GI inflammation.

Urinary eosinophilic protein X, atopy, and symptoms suggestive of allergic disease at 3 years of age.

BACKGROUND: Urinary eosinophilic protein X (U-EPX) measurement is easy to perform in children. However, its use for prediction, diagnosis, and monitoring of asthma and atopy is unclear. OBJECTIVE: We sought to investigate the relationship between U-EPX and clinical phenotypes suggestive of allergic diseases. METHODS: U-EPX measurement (RIA), respiratory questionnaires, and skin testing were completed at age 3 years in 903 children followed prospectively from birth. Specific airway resistance was measured in 503 currently asymptomatic children by using whole-body plethysmography during tidal breathing. RESULTS: Nonatopic children with wheezing or eczema had slightly increased U-EPX levels compared with nonatopic asymptomatic children. U-EPX levels (geometric mean EPX/creatinine ratio) were as follows: nonatopic asymptomatic children (n = 313), 61.3 microg/mmol (95% CI, 56.4-66.6 microg/mmol); nonatopic children with wheezing (n = 148), 71.2 microg/mmol (95% CI, 63.2-80.1 microg/mmol); nonatopic children with eczema (n = 90), 65.7 microg/mmol (95% CI, 56.7-76.2 microg/mmol); and nonatopic children with wheezing and eczema (n= 86), 79.7 microg/mmol (95% CI, 67.4-94.3 microg/mmol). Children who had persistent atopy early in life had significantly higher U-EPX levels at age 3 years (nonatopic at 1 and 3 years [n = 263], 63.4 microg/mmol [95% CI, 58.4-69.0 microg/mmol]; atopic at 1 but not 3 years [n = 24], 65.1 microg/mmol [95% CI, 43.8-96.7 microg/mmol]; nonatopic at 1 year and atopic at 3 years [n = 62], 90.0 microg/mmol [95% CI, 74.6-108.4 microg/mmol]; atopic at 1 and 3 years [n = 35], 111.5 microg/mmol [95% CI, 89.2-139.3 microg/mmol]; P <.002). Atopy alone and with wheezing, eczema, or both was associated with significantly increased U-EPX levels (P <.0001). Wheezing appeared to be associated with higher U-EPX levels compared with eczema in both atopic and nonatopic children. The highest U-EPX level was found in atopic children with a history of wheezing and eczema (P <.0001). There was no relationship between U-EPX level and lung function. CONCLUSION: U-EPX level reflects the presence of atopy and associated symptoms and might be useful for monitoring the progression of allergic disease.