Estimation of numbers of mature and immature neutrophils in blood by a novel, rapid and simple technology.

Counting numbers of blood neutrophils is one of the most common laboratory tests in modern clinical medicine. In this report, we have tested the idea that immunoassay of major constituents of mature neutrophils might serve as proxy of cell counting and allow the development of rapid and simple point-of-care tests. The procedure may also allow for the estimate of the state of maturity of the circulating blood cells. Immunoassays for myeloperoxidase (MPO) and lactoferrin (LF) were used to measure the respective protein in whole blood extracts of 275 unselected hospitalized patient and in 51 healthy controls and leukemia patients of which eight were followed before, during and after remission treatment. MPO was correlated to neutrophil counts in the unselected hospitalized population (r = 0.95, p <.0001). Huge variations were seen in whole blood extracts of patients with AML with very high MPO/LF ratios in half of the AML patients and in all three patients with APL. In extracts from patients with ALL no difference was found in the ratio as compared to healthy persons. The monitoring of AML patients during remission treatment showed intriguing patterns one of which suggested the possibility to monitor the myelopoietic activity in the bone marrow during the recovery phase. We show a novel and easy technology to count mature neutrophils in blood and also to monitor myeloid cell maturity in the blood as well as myelopoietic activity in the bone marrow. The technology lends itself to the development of a rapid and simple point-of-care test.

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.

Evaluation of the Meritas® BNP Test for Point-of-Care Testing.

BACKGROUND: BNP and NT-proBNP are widely used as rule-out tests for heart failure (HF) and they are frequently requested by primary care doctors. A point-of-care (POC) test would reduce the time to diagnosis for patients with suspected HF. The aim of the study was to evaluate a POC BNP test. METHODS: Plasma BNP results obtained with the Meritas® POC instrument (n = 82) were compared with the corresponding plasma BNP results analyzed on an Advia Centaur analyzer (Siemens Healthcare, Erlangen, Germany). RESULTS: The two methods showed concordant results with a Passing-Bablok correlation between the two methods: BNP(Meritas) = 1.00 x BNP(Siemens) + 1.09; r = 0.9773. CONCLUSIONS: The study show that the Meritas® BNP assay could be used in primary care permitting rapid BNP testing to rule out heart failure.

Fecal Biomarkers of Neutrophil and Eosinophil Origin Reflect the Response to Biological Therapy and Corticosteroids in Patients With Inflammatory Bowel Disease.

INTRODUCTION: Fecal calprotectin (FC) is a noninvasive tool for examining response to biologics in inflammatory bowel disease (IBD), but its performance in relation to other novel fecal markers of various cellular origins is unknown. METHODS: We performed a prospective multicenter cohort study and included patients with active IBD who provided a fecal sample at initiation of biological therapy. Levels of FC, myeloperoxidase (MPO), human neutrophil lipocalin (HNL), and eosinophil-derived neurotoxin (EDN) were analyzed and related to clinical remission status at 3 months. Changes in levels of markers at 3 months were calculated, and the impact of concomitant use of corticosteroids at baseline was estimated. RESULTS: In patients achieving clinical remission (n = 27), a decrease in levels of FC ( P = 0.005), MPO ( P < 0.001), HNL ( P < 0.001), and EDN ( P < 0.001) was observed, whereas no significant decrease was seen in patients not achieving remission (n = 39). There was a significant difference in the change in the level of MPO ( P = 0.01) and HNL ( P = 0.02) between patients achieving clinical remission and those who did not, but changes in FC and EDN could not differentiate between these groups. Patients with concomitant systemic corticosteroids at inclusion had lower levels of HNL ( P = 0.01) and EDN ( P < 0.001) at baseline, compared with patients without corticosteroids. DISCUSSION: Fecal MPO, HNL, and EDN are all promising biomarkers for assessing the treatment outcome of biologics in patients with IBD. Fecal levels of EDN and HNL are significantly affected by corticosteroids indicating a greater sensitivity to the effects of corticosteroids compared with levels of FC and MPO.

Elevated F-EDN correlates with mucosal eosinophil degranulation in patients with IBS-A possible association with microbiota?

Eosinophils have been linked to functional dyspepsia; however, less is known about their role in irritable bowel syndrome (IBS). This study tested the hypothesis of alterations in levels of fecal eosinophil-derived neurotoxin (F-EDN) and eosinophil density and degranulation within the colonic mucosa of IBS patients compared with healthy controls (HC). Colonic biopsies were collected from 37 IBS patients and 20 HC and analyzed for eosinophil numbers and local degranulation of eosinophil cationic protein (ECP) by histologic procedures. Fecal samples were collected for F-EDN and microbiota analysis. Differentiated 15HL-60 cells were used in vitro to investigate the direct effect of live bacteria on eosinophil activation measured by a colorimetric assay with o-phenylenediamine (OPD) substrate. We observed a higher number of eosinophils and increased extracellular ECP in the mucosa of IBS patients compared with HC. Moreover, F-EDN levels in IBS samples were elevated compared with HC and positively correlated to extracellular ECP. Metagenomic analysis showed significant correlations between bacterial composition and eosinophil measurements in both HC and IBS patients. In vitro experiments revealed an increased degranulation of 15HL-60 after stimulation with Salmonella typhimurium, Salmonella enterica, and Yersinia enterocolitica. To conclude, we could demonstrate alterations related to eosinophils in IBS, and, for the first time, a positive correlation between F-EDN levels and degranulated eosinophils in the colonic mucosa of IBS patients. Together our results suggest that eosinophils play a role in the pathophysiology of IBS and the mechanisms might be linked to an altered microbiota.

A multiplexed point-of-care assay for C-reactive protein and N-terminal pro-brain natriuretic peptide.

Several new plasma protein biomarkers have been associated with increased risk of cardiovascular events. It would be of great value if sets of these markers could be measured in a multiplexed format at point-of-care settings. A major challenge is the extremely wide concentration range in which different plasma biomarkers are present. Two promising biomarkers for cardiac risk prediction are C-reactive protein (CRP) and N-terminal pro-brain natriuretic peptide (NTproBNP). The concentrations of these markers can differ by more than six orders of magnitude. Here we present a chip-based multiplexed assay for CRP and NTproBNP. The high-concentration analyte, CRP, is analyzed in a competitive format, whereas the low-concentration analyte, NTproBNP, is analyzed in a sandwich format. This allows concurrent measurement of the two analytes in a single multiplexed assay. The dynamic ranges for the two assays were optimized to match the relevant serum concentration ranges; thus, no dilutions were needed. Both assays exhibit good precision (5-15% in the clinically relevant concentration ranges), and the limit of detection for the NTproBNP assay was 5 ng/L. Patient plasma samples were used for comparison with clinical methods, resulting in coefficients of determination (R(2)) of 0.9762 and 0.9606 for NTproBNP and CRP, respectively.

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.

HNL (Human Neutrophil Lipocalin) and a multimarker approach to the distinction between bacterial and viral infections.

OBJECTIVES: The distinction between bacterial and viral causes of acute infections is a major clinical challenge. In this report we investigate the diagnostic performance in this regard of nine candidate biomarkers together with HNL (Human Neutrophil Lipocalin). METHODS: Blood was obtained from patients with symptoms of infectious (n = 581). HNL was measured in whole blood (B-HNL) after pre-activation with the neutrophil activator fMLP or in plasma (P-HNL). Azurocidin also known as heparin-binding protein (HBP), Calprotectin, PMN-CD64, CRP (C-reactive protein), IP-10 (Interferon γ-induced Protein 10 kDa), PCT (Procalcitonin), TK1 (Thymidine kinase 1), TRAIL (TNF-related apoptosis-inducing ligand) were measured in plasma/serum. Area under the ROC (receiver operating characteristics) curve (AuROC) was used for the evaluation of the clinical performance of the biomarkers. RESULTS: Side-by-side comparisons of the ten biomarkers showed large difference in the AuROC with B-HNL being the superior biomarker (0.91, 95% CI 0.86-0.95) and with the other nine biomarkers varying from AuROC of 0.63-0.79. The combination of B-HNL with IP-10 and/or TRAIL increased the diagnostic performance further to AuROCs of 0.94-0.97. The AuROCs of the combination of CRP with IP-10 and/or TRAIL were significantly lower than combinations with B-HNL 0.87 (95% CI 0.83-0.91). CONCLUSION: The diagnostic performance of whole blood activated HNL was superior in the distinction between bacterial or viral infections. The addition of IP-10 and/or TRAIL to the diagnostic algorithm increased the performance of B-HNL further. The rapid analysis of HNL, reflecting bacterial infections, together with biomarkers reflecting viral infections may be the ideal combination of diagnostic biomarkers of acute infections.

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.

Human neutrophil lipocalin in fMLP-activated whole blood as a diagnostic means to distinguish between acute bacterial and viral infections.

UNLABELLED: The distinction between causes of acute infections is a major clinical challenge. Current biomarkers, however, are not sufficiently accurate. Human neutrophil lipocalin (HNL) in serum distinguishes acute infections with high accuracy, but in the emergency setting the assay time should be <15-20min, which excludes the use of serum samples. The aim was therefore to develop a novel rapid assay principle and test its clinical performance. METHODS: Serum and neutrophils obtained from 84 infected and 20 healthy subjects were used in the experimental study. 725 subjects (144 healthy controls and 581 patients with signs and symptoms of acute infections) were included in the clinical study. HNL was measured in EDTA-plasma by ELISA or in heparinized whole blood after fMLP activation by a prototype point-of-care assay. RESULTS: Increased release of HNL from neutrophils after activation with fMLP was seen already after 5 min incubation. The release of HNL from purified neutrophils after 15 min incubation with fMLP was significantly correlated to the HNL concentrations in serum obtained from the same patient (r = 0.74, p < 0.001). In the distinction between healthy controls and patients with bacterial infections, the areas under the ROC-curves were 0.95 (95% CI 0.91-0.97) and 0.88 (95% CI 0.84-0.91) for HNL in fMLP-activated whole blood and EDTA-plasma, respectively, (p < 0.001) and in the distinction between bacterial and viral infections 0.91 (95% CI 0.86-0.95) and 0.76 (95% CI 0.70-0.81), respectively (p < 0.001). CONCLUSION: The clinical performance of HNL in fMLP-activated whole blood was superior to HNL in EDTA-plasma and similar to HNL in serum. The procedure can be adopted for point-of-care testing with response times of <15 min.

Human Neutrophil Lipocalin as a Superior Diagnostic Means To Distinguish between Acute Bacterial and Viral Infections.

The distinction between causes of acute infections is a major clinical challenge. Current biomarkers, however, are not sufficiently accurate. Human neutrophil lipocalin (HNL) concentrations in serum or whole blood activated by formyl-methionine-leucine-phenylalanine (fMLP) were shown to distinguish acute infections of bacterial or viral cause with high accuracy. The aim was therefore to compare the clinical performance of HNL with currently used biomarkers. Seven hundred twenty-five subjects (144 healthy controls and 581 patients with signs and symptoms of acute infections) were included in the study. C-reactive protein (CRP), the expression of CD64 on neutrophils, procalcitonin (PCT), and blood neutrophil counts were measured by established techniques, and HNL concentrations were measured in whole-blood samples after activation with fMLP. All tested biomarkers were elevated in bacterial as opposed to viral infections (P < 0.001). CRP, PCT, and CD64 expression in neutrophils was elevated in viral infections compared to healthy controls (P < 0.001). In the distinction between healthy controls and patients with bacterial infections, the areas under the receiver operating characteristic (ROC) curves were >0.85 for all biomarkers, whereas for the distinction between bacterial and viral infections, only HNL concentration in fMLP-activated whole blood showed an area under the ROC curve (AUROC) of >0.90 and superior clinical performance. The clinical performance of HNL in fMLP-activated whole blood was superior to current biomarkers and similar to previous results of HNL in serum. The procedure can be adopted for point-of-care testing with response times of <15 min.

Purification and characterization of a 95-kDa protein--carcinoembryonic antigen-related cell adhesion molecule 8--from normal human granulocytes.

A 95-kDa protein was purified to homogeneity from granule extracts of normal human granulocytes. The column procedure consisted of Sephadex G-75, Mono-S cation exchange and Superdex HR 75 chromatography. The purified protein showed only one broad band at a molecular weight of 95 kDa when analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It reacted with polyclonal antibodies against carcinoembryonic antigen (CEA) and a specific monoclonal antibody against CD66b, but did not react with monoclonal antibodies against CD66acde and CD66c when analyzed by immunoblotting. The molecular weight of the protein shifted from 95 to 40 kDa on SDS-PAGE after deglycosylation. Tryptic peptide analysis by MALDI-Tof identified four peptides with spectra of m/z matching the expected tryptic peptides from a CGM6 gene product. Furthermore, the nanoelectrospray mass spectrometry (MS/MS) analysis of the two selected tryptic peptides of the protein revealed two amino acid sequences corresponding to residues 79-98 and 199-207 of the CGM6 gene product. Based on this, and also on the immunochemical data, it is concluded that the purified 95 kDa is identical to carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8) (nonspecific cross-reacting antigen (NCA)-95, CD67 and CD66b) and is a product of the CGM6 (W272) gene. We present, for the first time, a method for the purification of CEACAM8 from normal human granulocytes, which should be useful for further studies on its structure and functions. We also confirmed at the protein level that CEACAM8 is a product of the CGM6 (NCA-W272) gene.

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.

Detection of inflammatory markers in stools from patients with irritable bowel syndrome and collagenous colitis.

OBJECTIVE: Irritable bowel syndrome (IBS) and collagenous colitis (CC) share chronically recurring symptoms of altered bowel habits associated with abdominal pain or discomfort. The aims of the present study were to investigate whether inflammatory markers could be detected in faeces from patients with IBS and CC, and to elucidate whether such analyses could be used as non-invasive tools to distinguish between these disorders. MATERIAL AND METHODS: Stool samples were obtained from 18 patients with CC, 46 patients with IBS and 20 healthy controls (HC). Eosinophil protein X (EPX), myeloperoxidase (MPO), tryptase, interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha) were measured in supernatants from processed faeces using immunoassays. RESULTS: EPX levels were enhanced in faeces from CC patients (median 3.8 microg/g (0.47-16.2)) compared to patients with IBS (0.44 microg/g (0.25-1.8)), p<0.001, and HC (0.46 microg/g (0.21-1.3)), p<0.001. In addition, MPO was increased in CC patients (11.7 microg/g (2.0-124)) compared to IBS patients (1.7 microg/g (0.81-5.2)), p<0.01, and HC (2.5 microg/g (1.1-6.3)), p<0.05. Tryptase was found in 9/18 patients with CC, 6/46 with IBS and 1/19 HC. IL-1beta was only enhanced in 2/11 CC patients and TNFalpha was not detected in any sample. CONCLUSIONS: Increased levels of EPX, MPO and tryptase were observed in stools from collagenous colitis patients, whereas the levels in IBS patients did not differ from healthy controls. Our data suggest that faecal markers could be used as part of the clinical work-up to determine which patients should be biopsied and evaluated for collagenous colitis.

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.