One-Hour Esophageal String Test: A Nonendoscopic Minimally Invasive Test That Accurately Detects Disease Activity in Eosinophilic Esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE), a chronic food allergic disease, lacks sensitive and specific peripheral biomarkers. We hypothesized that levels of EoE-related biomarkers captured using a 1-hour minimally invasive Esophageal String Test (EST) would correlate with mucosal eosinophil counts and tissue concentrations of these same biomarkers. We aimed to determine whether a 1-hour EST accurately distinguishes active from inactive EoE or a normal esophagus. METHODS: In a prospective, multisite study, children and adults (ages 7-55 years) undergoing a clinically indicated esophagogastroduodenoscopy performed an EST with an esophageal dwell time of 1 hour. Subjects were divided into 3 groups: active EoE, inactive EoE, and normal esophageal mucosa. Eosinophil-associated protein levels were compared between EST effluents and esophageal biopsy extracts. Statistical modeling was performed to select biomarkers that best correlated with and predicted eosinophilic inflammation. RESULTS: One hundred thirty-four subjects (74 children, 60 adults) with active EoE (n = 62), inactive EoE (n = 37), and patient controls with a normal esophagus (n = 35) completed the study. EST-captured eosinophil-associated biomarkers correlated significantly with peak eosinophils/high-power field, endoscopic visual scoring, and the same proteins extracted from mucosal biopsies. Statistical modeling, using combined eotaxin-3 and major basic protein-1 concentrations, led to the development of EoE scores that distinguished subjects with active EoE from inactive EoE or normal esophagi. Eighty-seven percent of children, 95% of parents, and 92% of adults preferred the EST over endoscopy if it provided similar information. DISCUSSION: The 1-hour EST accurately distinguishes active from inactive EoE in children and adults and may facilitate monitoring of disease activity in a safe and minimally invasive fashion.

Epidermal expression of eotaxins and thymic stromal lymphopoietin in eosinophil rich dermatoses.

Eosinophils are seen in a number of dermatologic conditions. While the extent of their function in these diseases remains to be fully elucidated, pathogenic activity in bullous pemphigoid suggests a more significant role than previously thought. Several dermatoses have a fairly characteristic histologic morphology of eosinophil infiltration. We hypothesized that epidermal expression of eotaxins and TSLP would differ by disease, perhaps explaining the different histologic morphologies. We performed a retrospective study of eosinophil rich dermatoses to perform immunohistochemistry. We collected 49 specimens composed of bullous pemphigoid (n = 15), atopic dermatitis (n = 12), drug rash (n = 8), arthropod assault (n = 5), and non-bullous pemphigoid eosinophilic spongiosis (n = 5). We used lichen planus (n = 4) as a control for lymphocyte-mediated inflammation. TSLP was diffusely expressed in all epidermal samples, whereas eotaxins demonstrated a weaker staining. Eotaxins and TSLP demonstrated a gradient between basal and spinous keratinocytes. The correlation between overall basal keratinocyte and spinous keratinocyte staining of eotaxins and TSLP with the number of eosinophils demonstrated a significant correlation between eotaxin-1 (R = 0.404, P = 0.004), eotaxin-2 (R = 0.576, P < 0.001), and eotaxin-3 (R = 0.512, P < 0.001), but not TSLP (R = 0.164, P = 0.251). These remained significant after correcting for multiple comparisons. While we were unable to detect significant differences in epidermal expression of eotaxins and TSLP in various eosinophil rich dermatoses, we identified a significant correlation of spinous keratinocyte eotaxin staining with tissue eosinophilia. Our identification of a correlation of spinous keratinocyte eotaxin staining with tissue eosinophilia may provide insight into local eosinophil chemotaxis.

Nasal fluid release of eotaxin-3 and eotaxin-2 in persistent sinonasal eosinophilic inflammation.

BACKGROUND: The aim of the present study was to measure eotaxin-3 (CCL26) and eotaxin-2 (CCL24) in nasal lavage fluid of patients with different forms of chronic sinonasal eosinophilic inflammation to evaluate their role in the pathophysiology of nasal hypereosinophilia. METHODS: The study was an analytic cross-section study, level of evidence 3b. Patients (n = 80) with nasal hypereosinophilia were randomly recruited and grouped in the following categories: persistent allergic rhinitis (AR) (n = 25), nonallergic rhinitis with eosinophilia syndrome (NARES) (n = 30), and chronic rhinosinusitis with polyps (CRSwNP) (n = 25). Non-rhinitic volunteers (n = 20) were recruited as controls. CCL24 and CCL26 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) Quantikine Human Immunoassays (R&D Systems, Minneapolis, MN) in nasal lavage fluids. Differential cell counts were performed by microscopic cytological examination of nasal tissue scraped from the inferior turbinate. RESULTS: Mean CCL26 levels were significantly higher (p < 0.05) in AR and in NARES (132.0 pg/mL and 187.63 pg/mL, respectively) than in the control group (13.5 pg/mL); in patients with CRSwNP, CCL26 values were increased compared to controls even though the difference was not statistically significant (58.9 pg/mL vs 16.5 pg/mL). Mean CCL24 levels measured in AR, NARES, and CRSwNP were significantly increased (p < 0.05) compared to controls (96.7 pg/mL, 135.4 pg/mL, and 107.0 pg/mL, respectively, vs 32.2 pg/mL). Moreover, we observed a significant correlation between CCL24 and CCL26 levels, evaluating them intraindividually by Spearman's rank correlation test. Finally, a significant correlation was found between CCL24 and CCL26 levels and the percentage of eosinophilic infiltration of nasal mucosa. CONCLUSION: Our data suggest that CCL26 and CCL24 are likely involved in the pathogenesis of chronic nasal hypereosinophilia, with a complex cooperation and different involvement of the various members of eotaxin family. Further studies are necessary to better understand the actual physiopathologic mechanism, possible clinical relevance, and therapeutic implications.

Intraepithelial lymphocyte eotaxin-2 expression and perineural mast cell degranulation differentiate allergic/eosinophilic colitis from classic IBD.

OBJECTIVES: Allergic colitis shows overlap with classic inflammatory bowel disease (IBD). Clinically, allergic colitis is associated with dysmotility and abdominal pain, and mucosal eosinophilia is characteristic. We thus aimed to characterise mucosal changes in children with allergic colitis compared with normal tissue and classic IBD, focusing on potential interaction between eosinophils and mast cells with enteric neurones. METHODS: A total of 15 children with allergic colitis, 10 with Crohn disease (CD), 10 with ulcerative colitis (UC), and 10 histologically normal controls were studied. Mucosal biopsies were stained for CD3 T cells, Ki-67, eotaxin-1, and eotaxin-2. Eotaxin-2, IgE, and tryptase were localised compared with mucosal nerves, using neuronal markers neurofilament protein, neuron-specific enolase, and nerve growth factor receptor. RESULTS: Overall inflammation was greater in patients with CD and UC than in patients with allergic colitis. CD3 T-cell density was increased in patients with allergic colitis, similar to that in patients with CD but lower than in patients with UC, whereas eosinophil density was higher than in all other groups. Eotaxin-1 and -2 were localised to basolateral crypt epithelium in all specimens, with eotaxin-1+ lamina propria cells found in all of the colitis groups. Eotaxin-2+ intraepithelial lymphocyte (IEL) density was significantly higher in allergic colitis specimens than in all other groups. Mast cell degranulation was strikingly increased in patients with allergic colitis (12/15) compared with that in patients with UC (1/10) and CD (0/1). Tryptase and IgE colocalised on enteric neurons in patients with allergic colitis but rarely in patients with IBD. CONCLUSIONS: Eotaxin-2+ IELs may contribute to the periepithelial eosinophil accumulation characteristic of allergic colitis. The colocalisation of IgE and tryptase with mucosal enteric nerves is likely to promote the dysmotility and visceral hyperalgesia classically seen in allergic gastrointestinal inflammation.

Eosinophil degranulation is more important than eosinophilia in identifying asthma in chronic cough.

OBJECTIVE: We investigated whether eosinophil degranulation is a distinctive feature of asthma and can distinguish between chronic cough patients with asthma and those without. METHODS: Thirty-seven patients, with a chronic cough for more than 1 month, and nine normal individuals (controls) were enrolled. Subjects were divided into two groups: one group with asthma and positive bronchial hyperresponsiveness (BHR) (Asthma group, n = 18) and the other group without asthma and negative BHR (Non-Asthma group, n = 19). From induced sputum, total cell counts and differentials were determined. Myeloperoxidase levels were measured by Enzyme-Linked Immunosorbent Assay (ELISA), and eosinophil-derived neurotoxin (EDN) and major basic protein (MBP) levels were measured by radioimmunoassay. RESULTS: The percentage of sputum eosinophils was increased in the Asthma (p < .001) and Non-Asthma (p < .05) groups compared with the Control group and when comparing the Asthma and Non-Asthma (p < .001) groups. Sputum EDN and MBP levels were increased in the Asthma group compared with the Non-Asthma (p < .05 and p < .05, respectively) and Control groups (p < .05 and p = .055, respectively). However, EDN and MBP levels were not increased in the Non-Asthma group compared with the Control group. The percentage of sputum eosinophils in the Asthma group correlated positively with sputum EDN (Rs = 0.921, p < .001) and MBP (Rs = 0.882, p < .0001) levels and negatively with maxΔFEV(1) (Rs = -0.501, p < .05) (FEV(1), forced expiratory volume in 1 second). Unexpectedly, the percentage of eosinophils in the Non-Asthma group did not correlate significantly with any of these markers. Increased EDN and MBP levels and significant correlations between the percentage of eosinophils and EDN and MBP were only observed in asthma patients. CONCLUSIONS: These findings suggest that eosinophil degranulation is more important than eosinophilia in identifying asthma.

Nasal lavage CCL24 levels correlate with eosinophils trafficking and symptoms in chronic sino-nasal eosinophilic inflammation.

OBJECTIVES: The aim of our study was to measure CCL24 (eotaxin-2) levels in nasal lavage fluid of patients with different forms of sinonasal chronic eosinophilic inflammation to verify the relationship with nasal hypereosinophilia and symptoms. METHODS: Patients with nasal hypereosinophilia were randomly recruited and grouped in persistent allergic rhinitis, non-allergic rhinitis with eosinophilia syndrome (NARES) and chronic rhinosinusitis with polyps. Non rhinitic volunteers were recruited as controls. CCL24 concentration was measured by `Quantikine Human CCL24 Immunoassay`. Differential cell counts were performed by microscopic cytological examination of nasal tissue scraped by inferior turbinate. RESULTS: CCL24 levels measured in patient groups were significantly higher compared to control group with the highest levels in NARES patients. Eotaxin- 2 levels were significantly correlated to severity of symptoms and to the percentage of eosinophils in nasal tissue. CONCLUSIONS: We revealed high levels of CCL24 in all patient groups showing a significant correlation with the degree of eosinophilia and clinical symptoms. A prolonged accumulation of CCL24 inside the nasal mucosa may sustain the process of unspecific self-perpetuating eosinophil recruitment pathognomonic of these patients.

[Allergy related factors in tears of patients with vernal keratoconjunctivitis undergoing topical 0.1% tacrolimus treatment].

PURPOSE: To investigate, using tear fluid analysis, the effects of topical 0.1% tacrolims therapy on the pathophysiology of vernal keratoconjunctivitis (VKC). SUBJECTS AND METHODS: Subjects were 6 eyes of 6 patients with VKC who underwent topical 0.1% tacrolims treatment twice a day and 5 eyes of 5 healthy volunteers as a control. Using the filter paper method, the tear fluid of the patients was sampled both before and 4 +/- 2 weeks after the treatment and once from the control subjects. Eosinophil cationic protein (ECP) in the tears was examined by the chemiimmunoluminescent enzyme immunoassay method and the chemokine profile of the tears was analyzed using an antibody-array. RESULTS: In terms of the chemokine profile, growth related oncogene (GRO) -alpha, eotaxin-2 and thymus and activation-regulated chemokine (TARC) in the VKC were elevated compared with those in the controls, but they decreased significantly after the treatment (p<0.05). ECP concentrations in the tears were 3092 +/- 1658 ng/ml (average +/- S. D.) for the pretreatment base-line and 464 +/- 775 for the posttreatment. ECP values for the pre-treatment time were statistically significantly higher than those for the post-treatment in 5 patients (p<0.05). CONCLUSION: Topical tacrolims treatment of VKC can suppress allergic inflammation associated chemokines such as eotaxin-2 and TARC.

Smoking affects eotaxin levels in asthma patients.

BACKGROUND: Chronic airway inflammation is most important pathological finding in asthma. Cigarette smoking may modify type of inflammation as well as may influence disease severity and response to the treatment. OBJECTIVE: Thus the aim of this study was to investigate whether cigarette smoking may have an influence on the levels of eotaxin-1, eotaxin-2, eotaxin-3 and IL-5 in patients with stable mild/moderate asthma. METHODS: 45 steroid naive asthmatics (mean age: 55.2 +/- 2.2 yrs) and 23 "healthy" smokers and non-smokers control subjects (mean age: 54.4 +/- 9.7 yrs) were investigated. Asthmatics were divided into two subgroups according to their smoking histories: asthmatic smokers (n = 19) who currently smoke and have a history of > 10 pack-years and asthmatic never-smokers (n = 26). BAL and induced sputum were performed. Cytospins of induced sputum and BAL were stained with May-Grunwald-Giemsa for differential cell counts. Eotaxin-1, eotaxin-2, eotaxin-3 and IL-5 concentrations in serum, sputum and BAL supernatant was measured using a commercial ELISA kit. RESULTS: In sputum supernatant from asthma smokers was significantly higher concentration of eotaxin-1 than in non-smokers asthmatics (203.4 +/- 10.0 vs. 140.2 +/- 9.5 respectively, p < 0.05). In non-smokers asthma patients levels of BAL eotaxin-1 strongly related to percent and absolute numbers of BAL eosinophils and neutrophils (Rs = 0.737 and Rs = 0.514 respectively, p < 0.05). The number and percent of sputum neutrophils and eosinophils, obtained from smokers asthmatics, significantly correlated with eotaxin-2 concentration in sputum supernatant (Rs = 0.58 and Rs = 0.75 respectively, p < 0.05). IL-5 levels in the serum and sputum from asthmatic never-smokers were significantly higher than they were from asthmatic smokers and "healthy" smokers. Asthmatic never-smokers showed a significantly higher amount of IL-5 in serum and sputum than the asthmatic smokers showed. CONCLUSIONS: This study showed the elevated levels of sputum eotaxin-1 as well as serum, sputum and BAL eotaxin-2 in asthmatic smokers without a significant increase of eosinophils compared to asthmatic never-smokers. The eotaxin concentrations were related not only with number of eosinophils but also with the number of neutrophils in all the studied tissue compartments. The data herein permits a suggestion that smoking may influence change in asthmatic airway inflammation by stimulating the production of eotaxins.

Eotaxin-1, -2, and -3 immunoreactivity and protein concentration in the nasal polyps of eosinophilic chronic rhinosinusitis patients.

OBJECTIVES/HYPOTHESIS: Eosinophilic chronic rhinosinusitis (CRS) is characterized by the accumulation of numerous eosinophils in the sinus mucosa and nasal polyps, which are frequently difficult to control, even with surgery. The present study was designed to evaluate the expression and localization of eotaxins, which are well known to be potent and selective chemoattractants for eosinophils in CRS. STUDY DESIGN: Randomized study. METHODS: The patients were classified into eosinophilic and noneosinophilic groups. Histopathological profiles of the nasal polyp were observed with hematoxylin-eosin staining. Eotaxin-1, -2, and -3 were immunohistochemically stained in the nasal polyps. Furthermore, the protein content of eotaxin subtypes inside the nasal polyp and sinus effusion was measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: In the nasal polyps, immunoreactivities of the eotaxin subfamily, eotaxin-1, -2, and -3, were noted in most of the infiltrating eosinophils, as well as in other inflammatory cells, epithelial cells, and endothelial cells. Compared with noneosinophilic CRS groups, eosinophilic CRS groups had a significant expression of eotaxins in their eosinophils. The eotaxin concentrations of nasal polyp and sinus effusion as measured by ELISA were significantly increased in the eosinophilic CRS group compared to the noneosinophilic CRS group. CONCLUSIONS: The present findings suggest that enhanced eotaxin family production by eosinophils results in the recruitment of eosinophils into the tissue by a self-amplifying process. Laryngoscope, 2009.

Protein microarray analysis in patients with asthma: elevation of the chemokine PARC/CCL18 in sputum.

BACKGROUND: Microarray technology offers a new opportunity to gain insight into global gene and protein expression profiles in asthma. To identify novel factors produced in the asthmatic airway, we analyzed sputum samples by using a membrane-based human cytokine microarray technology in patients with bronchial asthma (BA). METHODS: Induced sputum was obtained from 28 BA subjects, 20 nonasthmatic atopic control (AC) subjects, and 38 nonasthmatic nonatopic normal control (NC) subjects. The microarray samples of subjects were randomly selected from nine BA subjects, three AC subjects, and six NC subjects. Sputum supernatants were analyzed using a custom human cytokine array (RayBio Custom Human Cytokine Array; RayBiotech; Norcross, GA) designed to analyze 79 specific cytokines simultaneously. The levels of growth-regulated oncogene (GRO)-alpha, eotaxin-2, and pulmonary and activation-regulated chemokine (PARC)/CCL18 were measured by sandwich enzyme-linked immunosorbent assays (ELISAs), and eosinophil-derived neurotoxin (EDN) was measured by radioimmunoassay. RESULTS: By microarray, the signal intensities for GRO-alpha, eotaxin-2, and PARC were significantly higher in BA subjects than in AC and NC subjects (p = 0.036, p = 0.042, and p = 0.033, respectively). By ELISA, the sputum PARC protein levels were significantly higher in BA subjects than in AC and NC subjects (p < 0.0001). Furthermore, PARC levels correlated significantly with sputum eosinophil percentages (r = 0.570, p < 0.0001) and the levels of EDN (r = 0.633, p < 0.0001), the regulated upon activation, normal T cell expressed and secreted cytokine (r = 0.440, p < 0.001), interleukin-4 (r = 0.415, p < 0.01), and interferon-gamma (r = 0.491, p < 0.001). CONCLUSIONS: By a nonbiased screening approach, a chemokine, PARC, is elevated in sputum specimens from patients with asthma. PARC may play important roles in development of airway eosinophilic inflammation in asthma.