Searching for Noninvasive Predictors of the Diagnosis and Monitoring of Eosinophilic Esophagitis-The Importance of Biomarkers of the Inflammatory Reaction Involving Eosinophils.

BACKGROUND: Invasive and costly endoscopic diagnosis is obligatory for the diagnosis and monitoring of eosinophilic esophagitis (EoE). This study aims to evaluate the usefulness of serum biomarkers involved in eosinophil-mediated inflammation in the management of EoE. METHODS: A prospective cohort study was conducted in 58 patients with dysphagia. Each participant completed a health questionnaire, underwent esophagogastroduodenoscopy with esophageal biopsy for histopathological examination and assessment of total, inflammatory and fibrostenotic Eosinophilic Esophagitis Reference Score (EREFS). Serum levels of interleukin 5 (IL-5), interleukin 13 (IL-13), transforming growth factor ß1 (TGF-ß1), major basic protein (MBP), and eotaxin 3 were determined by enzyme immunoassays. Total of 16 patients meeting the histological criteria for EoE were treated with proton pump inhibitors for 8 weeks, and then the same diagnostics was performed again. RESULTS: Statistically significantly higher concentrations of MBP and TGF-ß1 were demonstrated in the group of patients with EoE, while MBP and eotaxin 3 correlated with the peak eosinophil count (PEC). Baseline MBP levels and eotaxin 3 after treatment significantly positively correlated with EREFS. There was a negative correlation between IL-13 and fibrostenotic EREFS. Additionally, after treatment, a negative correlation TGF-ß1 was noted with the inflammatory EREFS and a positive correlation with the fibrostenotic EREFS. CONCLUSIONS: The potential role of MBP in predicting the diagnosis of EoE, eotaxin 3 in predicting the advancement and correlation of IL-13 and TGF-ß1 in differentiating the inflammatory and fibrotic course of the disease may facilitate the management and individualization of EoE therapy.

Development and application of novel immunoassays for eosinophil granule major basic proteins to evaluate eosinophilia and myeloproliferative disorders.

BACKGROUND: During eosinophil differentiation, the granule eosinophil major basic protein 1 (eMBP1) is synthesized as a 32-kDa precursor form, referred to as proMBP1, which is processed into the 14-kDa mature form of eMBP1. The prevalence of these two forms of MBP1 in most pathological conditions remains unknown. OBJECTIVE: To develop the immunoassays that differentiate mature eMBP1 and proMBP1 and apply them to analyze their levels in biological fluids from patients with eosinophilia and hematologic disorders. METHODS: We produced a series of monoclonal antibodies and selected pairs capable of discriminating between the two molecular forms of eMBP1. Radioimmunoassay (RIA) was performed to simultaneously quantitate the levels of mature eMBP1 and proMBP1 in secretions from patients with chronic rhinosinusitis (CRS) and sera from patients with hypereosinophilic syndrome (HES) and other myeloproliferative disorders. RESULTS: The novel immunoassays possessed less than 1% crossreactivity between mature eMBP1 and proMBP1. Mature eMBP1, but not proMBP1, was found in nasal secretions of CRS patients. In contrast, elevated serum levels of mature eMBP1 and proMBP1 were observed in approximately 60% and 90% of HES patients, respectively, with proMBP1 present in greater quantities than mature eMBP1. Patients with several myeloproliferative disorders also showed high serum levels of proMBP1 while mature eMBP1 remained at basal levels. CONCLUSION: The novel immunoassays successfully differentiated mature eMBP1 and proMBP1 in human biological fluids. Further studies addressing the clinical correlates of these assays will help to develop biomarkers to diagnose and monitor patients with eosinophilia and myeloproliferative disorders.

The Effect of Helminths on Granulocyte Activation: A Cluster-Randomized Placebo-Controlled Trial in Indonesia.

BACKGROUND: Eosinophils are a prominent cell type in the host response to helminths, and some evidence suggests that neutrophils might also play a role. However, little is known about the activation status of these granulocytes during helminth infection. METHODS: We analyzed the expression of eosinophil and neutrophil activation markers in peripheral blood by flow cytometry and measured serum levels of eosinophil granule proteins in 300 subjects residing in an area endemic for soil-transmitted helminths (STH). The data generated are on samples before and after 1 year of 3-monthly albendazole treatment. RESULTS: Anthelmintic treatment significantly reduced the prevalence of STH. While eosinophil numbers were significantly higher in STH-infected compared to uninfected subjects and significantly decreased following albendazole treatment, there was no effect exerted by the helminths on either eosinophil nor neutrophil activation. Although at baseline eosinophil granule protein levels were not different between STH-infected and uninfected subjects, treatment significantly reduced the levels of eosinophil-derived neurotoxin (EDN) in those infected at baseline. CONCLUSIONS: These results show that besides decreasing eosinophil numbers, anthelmintic treatment does not significantly change the activation status of eosinophils, nor of neutrophils, and the only effect seen was a reduction in circulating levels of EDN. CLINICAL TRIALS REGISTRATION: http://www.isrctn.com/ISRCTN75636394.

Heightened Systemic Levels of Neutrophil and Eosinophil Granular Proteins in Pulmonary Tuberculosis and Reversal following Treatment.

Granulocytes are activated during Mycobacterium tuberculosis infection and act as immune effector cells, and granulocyte responses are implicated in tuberculosis (TB) pathogenesis. Plasma levels of neutrophil and eosinophil granular proteins provide an indirect measure of degranulation. In this study, we wanted to examine the levels of neutrophil and eosinophil granular proteins in individuals with pulmonary tuberculosis (PTB) and to compare them with the levels in individuals with latent TB (LTB). Hence, we measured the plasma levels of myeloperoxidase (MPO), neutrophil elastase, proteinase 3, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPX) in these individuals. Finally, we also measured the levels of all of these proteins in PTB individuals following antituberculosis treatment (ATT). Our data reveal that PTB individuals are characterized by significantly higher plasma levels of MPO, elastase, proteinase 3, as well as MBP and EDN in comparison to those in LTB individuals. Our data also reveal that ATT resulted in the reversal of all of these changes, indicating an association with TB disease. Finally, our data show that the systemic levels of MPO and proteinase 3 can significantly discriminate PTB from LTB individuals. Thus, our data suggest that neutrophil and eosinophil granular proteins could play a potential role in the innate immune response and, therefore, the pathogenesis of pulmonary TB.

Eosinophil Cationic Protein (ECP), a predictive marker of bullous pemphigoid severity and outcome.

Bullous Pemphigoid (BP) is an inflammatory rare autoimmune bullous dermatosis, which outcome cannot be predicted through clinical investigations. Eosinophils are the main immune infiltrated cells in BP. However, the release of Major Basic Protein (MBP), Eosinophil Derived Neurotoxin (EDN), and Eosinophil Cationic Protein (ECP) upon eosinophil activation has still not been evaluated with respect to BP development. MBP, EDN and ECP were measured by ELISA in serum (n = 61) and blister fluid (n = 20) of patients with BP at baseline, and in serum after 2 months of treatment (n = 41). Eosinophil activation in BP patients was illustrated at baseline by significantly higher MBP, EDN and ECP serum concentrations as compared with control subjects (n = 20), but without distinction according to disease severity or outcome. EDN and ECP values were even higher in the blister fluids (P < 0.01 and P < 0.05, respectively), whereas MBP values were lower (P < 0.001). ECP serum concentration decreased after 60 days of treatment in BP patients with ongoing remission but not in patients who later relapsed (P < 0.05). A reduction of at least 12.8 ng/mL in ECP concentrations provided a positive predictive value for remission of 81%, showing that ECP serum variation could be a useful biomarker stratifying BP patients at risk of relapse.

Pregnancy-associated plasma protein-A (PAPP-A) and the proform of the eosinophil major basic protein (ProMBP) are associated with increased risk of death in heart failure patients.

Risk stratification and patient management in heart failure (HF) is difficult due to the unpredictable progression of the disease, necessitating the development of reliable diagnostic biomarkers to facilitate decision-making in clinical practice. Pregnancy-associated plasma protein-A (PAPP-A) is a marker of arteriosclerotic heart disease. PAPP-A is a serum protease, which is involved in the insulin-like growth factor 1 (IGF-1) axis where it is inhibited by the proform of the eosinophil major basic protein (proMBP). In this study, we evaluated serum PAPP-A and proMBP as long-term prognostic biomarkers of all-cause mortality in HF. Serum PAPP-A and proMBP concentrations were determined in 683 patients with NYHA III-IV HF recruited in the EchoCardiography and Heart Study (ECHOS) in Denmark. The mean age of the patients (73% male) was 70 at admission. During 7 years of follow-up, 516 patients died. In univariate analysis, both PAPP-A and proMBP, divided into quartiles, showed significant association with mortality. Using a Cox proportional hazard model, hazard ratios for continuous values of PAPP-A and proMBP were HR = 1.42 (CI = 1.23-1.64, p < 0.0001) and HR = 1.36 (CI = 1.22-1.51, p <0.0001), respectively. However, neither PAPP-A nor proMBP were significant independent predictors when the model included age, gender, brain-type natriuretic peptide, medical history of HF, ischemic heart disease, chronic obstructive pulmonary disease, and diabetes mellitus. In conclusion, high levels of PAPP-A and proMBP are associated with increased risk of death from all causes in HF and are potential prognostic markers of adverse outcomes in HF patients.

Utility of a Noninvasive Serum Biomarker Panel for Diagnosis and Monitoring of Eosinophilic Esophagitis: A Prospective Study.

OBJECTIVES: Noninvasive biomarkers would be valuable for diagnosis and monitoring of eosinophilic esophagitis (EoE). The aim of this study was to determine the utility of a panel of serum biomarkers for the diagnosis and management of EoE. METHODS: We conducted a prospective cohort study of consecutive adults undergoing outpatient esophagogastroduodenoscopy. Incident cases of EoE were diagnosed per consensus guidelines; controls had gastroesophageal reflux disease (GERD) or dysphagia and did not meet the EoE criteria. EoE cases were treated with topical steroids and had repeat endoscopy. Pre- and post-treatment serum samples were analyzed in a blinded manner for interleukin (IL)-4, IL-5, IL-6, IL-9, IL-13, transforming growth factor (TGF)-α, TGF-ß, tumor necrosis factor-α, eotaxin-1, -2, and -3, thymic stromal lymphopoietin (TSLP), major basic protein, and eosinophil-derived neurotoxin. Cases and controls were compared at baseline, and pre- and post-treatment assays were compared in cases. RESULTS: A total of 61 incident EoE cases and 87 controls were enrolled; 51 EoE cases had post-treatment serum analyzed. There were no significant differences in any of the biomarkers between EoE cases and controls at baseline. IL-13 and eotaxin-3 for cases and controls were 85 ± 160 vs. 43 ± 161 pg/ml (P=0.12) and 41 ± 159 vs. 21 ± 73 (P=0.30). There were no significant differences in assay values among cases before and after treatment. There were also no differences after stratification by atopic status or treatment response. CONCLUSIONS: A panel of inflammatory factors known to be associated with EoE pathogenesis were not increased in the serum, nor were they responsive to therapy. None of these biomarkers are likely candidates for a serum test for EoE. Histologic analysis for diagnosis and management of EoE continues to be necessary, and novel, less invasive, biomarkers are needed.

Longitudinal changes in C-reactive protein, proform of eosinophil major basic protein, and pregnancy-associated plasma protein-A during weight changes in obese children.

BACKGROUND: Childhood obesity is associated with several complications, including cardiovascular comorbidity. Several biomarkers, such as high-sensitive C-reactive protein (hs-CRP), proform of eosinophil major basic protein (Pro-MBP) and pregnancy associated plasma protein-A (PAPP-A), have equally been linked to increased cardiovascular susceptibility. This study investigates these biomarkers during weight loss and regain in obese children. MATERIALS AND METHODS: A longitudinal study during a 12-week weight loss program with a 28 months follow-up was conducted. Anthropometrics and plasma concentrations of hs-CRP, Pro-MBP, and PAPP-A were measured at baseline; at days 14, 33 and 82 during weight loss; and at months 10, 16, and 28 during follow-up. RESULTS: Fifty-three boys and 62 girls aged 8-15 years with a median body mass index (BMI) standard deviation score (SDS) at baseline of 2.78 (boys), and 2.70 (girls) were included. Ninety children completed the weight loss program and 68 children entered the follow-up program. Pro-MBP and PAPP-A, but not hs-CRP, exhibited individual-specific levels (tracking) during weight loss and regain. The PAPP-A/Pro-MBP correlation was strong, whereas the hs-CRP/PAPP-A correlation was weak during weight fluctuations. CONCLUSION: Hs-CRP changes reflect weight changes. PAPP-A and Pro-MBP exhibited tracking during weight perturbations and may contribute as early risk markers of cardiovascular susceptibility.

Biology of pregnancy-associated plasma protein-A in relation to prenatal diagnostics: an overview.

Pregnancy-associated plasma protein-A (PAPP-A) is highly efficient as a serum marker in first-trimester screening for chromosomal abnormalities. Furthermore, there is increasing evidence that low levels of PAPP-A in the first trimester are associated with adverse pregnancy outcomes such as preterm delivery, intrauterine growth retardation, preeclampsia, and stillbirth. PAPP-A is a glycoprotein, produced in the placenta, and it is present in the maternal circulation in increasing concentrations during pregnancy. By means of its proteolytic activity, PAPP-A functions as a regulatory protein in the insulin-like growth factor system, known to be important for placental formation and regulation of fetal growth. This overview describes aspects of biochemistry, synthesis, and biological functions of PAPP-A, with a focus on information of importance to clinicians. The clinical applications of PAPP-A are summarized, and new insights regarding the analyses of PAPP-A discussed.

Free vs total pregnancy-associated plasma protein A (PAPP-A) as a predictor of 1-year outcome in patients presenting with non-ST-elevation acute coronary syndrome.

BACKGROUND: The free fraction of pregnancy-associated plasma protein A (FPAPP-A) was found to be the PAPP-A form released to the circulation in acute coronary syndrome (ACS). We estimated the prognostic value of FPAPP-A vs total PAPP-A (TPAPP-A) concentrations in forecasting death and nonfatal myocardial infarction (combined endpoint) in patients with non-ST-elevation ACS. METHODS: We recruited 267 patients hospitalized for symptoms consistent with non-ST-elevation ACS and followed them for 12 months. FPAPP-A, TPAPP-A, C-reactive protein (CRP), and cardiac troponin I (cTnI) were measured at admission; cTnI was also measured at 6-12 h and 24 h. Because of the recently shown interaction between PAPP-A and heparin, we excluded patients treated with any heparin preparations before the admission blood sampling. RESULTS: During the follow-up, 57 (21.3%) patients met the endpoint (22 deaths and 35 nonfatal myocardial infarctions). According to FPAPP-A (<1.27, 1.27-1.74, >1.74 mIU/L) and TPAPP-A (<1.98, 1.98-2.99, >2.99 mIU/L) tertiles, this endpoint was met by 12 (13.5%), 18 (20.2%), 27 (30.3%) (P = 0.02), and 17 (19.1%), 17 (19.1%), 23 (25.8%) (P = 0.54) patients, respectively. After adjusting for age, sex, diabetes, previous myocardial infarction, and ischemic electrocardiogram (ECG) findings, FPAPP-A >1.74 mIU/L [risk ratio (RR) 2.0; 95% CI 1.0-4.1, P = 0.053), increased cTnI, and CRP >/=2.0 mg/L were independent predictors of an endpoint. The prognostic performance of TPAPP-A was inferior to that of FPAPP-A. CONCLUSIONS: FPAPP-A seems to be superior as a prognostic marker compared to TPAPP-A, giving independent and additive prognostic information when measured at the time of admission in patients hospitalized for non-ST-elevation ACS.

First trimester maternal serum pregnancy-specific beta-1-glycoprotein (SP1) as a marker of adverse pregnancy outcome.

OBJECTIVE: To establish the first trimester levels of pregnancy-specific beta-1-glycoprotein (SP1) in pregnancies with adverse outcome. Furthermore, to determine the screening performance for adverse outcome using SP1 alone and in combination with other first trimester markers including proMBP and PAPP-A. METHODS: A case-control study was conducted in a primary hospital setting. The SP1 concentration was measured in first trimester maternal serum in pregnancies with small-for-gestational age fetuses (SGA) (n = 150), spontaneous preterm delivery (n = 88), preeclampsia (n = 40) and in controls (n = 500). Concentrations were converted to multiples of the median (MoM) in controls and groups were compared using Mann-Whitney U-test. Logistic regression analysis was used to determine significant factors for predicting adverse pregnancy outcome. Screening performance was assessed using receiver operating characteristic (ROC) curves. RESULTS: The SP1 MoM median was significantly reduced in cases with SGA (0.76 MoM, p < 0.0005) and spontaneous preterm delivery (0.77 MoM, p < 0.0005) whereas no alteration was found in cases with preeclampsia (0.94 MoM, p = 0.723). A significant correlation (r = 0.217) between log(10)(SP1 MoM) and the birth weight percentile was found in the SGA group. Screening performance was only slightly improved when SP1 was combined with PAPP-A or proMBP. CONCLUSION: SP1 is a first trimester maternal serum marker of SGA and preterm delivery.

Clinical correlates of serum pro-major basic protein in a spectrum of eosinophilic disorders and myelofibrosis.

BACKGROUND/AIMS: Eosinophil cytoplasmic granules contain major basic protein (MBP), which is primarily translated from its precursor, proMBP. In this study, we evaluated the plasma level of proMBP in eosinophilic and chronic myeloproliferative disorders (MPN). METHODS: The levels of plasma proMBP were measured by radioimmunoassay in 25 healthy controls, 23 patients with systemic mastocytosis (SM), 11 patients with idiopathic eosinophilia (IE) and a cohort of 170 patients with MPN which included 76 patients with de novo myelofibrosis, 42 with polycythemia vera (PV), 17 with postpolycythemic myeloid metaplasia (Post-PV MF), 21 with essential thrombocythemia (ET) and 14 with postthrombocythemic myeloid metaplasia (Post-ET MF). RESULTS: The plasma proMBP level was significantly higher in patients with SM with eosinophilia (p < 0.001), IE (p < 0.001) and MPN with eosinophilia (p = 0.002) than in healthy controls. The median proMBP level of Post-PV MF and Post-ET MF patients was significantly higher than in PV and ET patients (p < 0.001 and p = 0.009, respectively). In IE patients, elevated proMBP was significantly correlated with the presence of splenomegaly (p < 0.05). In 76 de novo myelofibrosis patients, the proMBP level was correlated with spleen size and the presence of hypercatabolic symptoms. CONCLUSION: The significantly elevated levels of proMBP in myelofibrosis patients implies that proMBP could be an important stromal cytokine in bone marrow fibrosis.

Prognostic value of circulating pregnancy-associated plasma protein-A (PAPP-A) and proform of eosinophil major basic protein (pro-MBP) levels in patients with chronic stable angina pectoris.

BACKGROUND: Pregnancy-associated plasma protein-A (PAPP-A) concentrations predict outcome in patients with acute coronary syndromes. PAPP-A levels and PAPP-A/pro-MBP ratio are increased in chronic stable angina (CSA) patients with complex coronary artery stenoses. Little is known however, about the long-term prognostic value of PAPP-A and pro-MBP in "real-life" CSA patients. We sought to assess whether PAPP-A, the proform of eosinophil major basic protein (pro-MBP) and PAPP-A/pro-MBP levels predict long-term all-cause mortality in patients with CSA. METHODS: We recruited 663 consecutive patients (169 women [25.5%]; mean age 62.9+/-9.7 years) undergoing routine diagnostic coronary angiography. Samples for PAPP-A and pro-MBP were taken at study entry. Patients were followed for a median of 8.8 years (interquartile range 3 - 10.6 years). RESULTS: 106 patients (16%) died during follow-up. On a Cox proportional hazards model, increased PAPP-A concentration (>4.8 mIU/L) was an independent predictor of the occurrence of all-cause mortality (HR 1.953, 95% CI 1.135-3.36, p=.016). Neither pro-MBP nor PAPP-A/pro-MBP ratio were markers of all-cause mortality (p=.45 and .54, respectively). CONCLUSIONS: High PAPP-A levels (>4.8 mIU/L) showed an association with all-cause mortality during long-term follow-up in patients with CSA.

Immunofluorometric point-of-care assays for the detection of acute coronary syndrome-related noncomplexed pregnancy-associated plasma protein A.

BACKGROUND: We recently reported that the pregnancy-associated plasma protein A (PAPP-A) form specifically related to acute coronary syndromes (ACS) is not complexed with the proform of eosinophil major basic protein (proMBP). The aim of this study was to develop rapid point-of-care immunoassays for the measurement of the noncomplexed PAPP-A. METHODS: We developed immunofluorometric noncompetitive dry-reagent assays for total PAPP-A with 2 PAPP-A subunit-specific monoclonal antibodies and for PAPP-A/proMBP complex with 1 PAPP-A subunit-specific antibody and 1 proMBP subunit-specific antibody. The concentration of noncomplexed PAPP-A was determined as the difference of the results obtained with the 2 assays. RESULTS: The assays were linear from 0.5 to 300 mIU/L. The analytical detection limit and functional detection limit (CV <20%) were 0.18 mIU/L and 0.27 mIU/L for total PAPP-A assay and 0.23 mIU/L and 0.70 mIU/L for PAPP-A/proMBP assay, respectively. The total assay imprecisions were <10%, and recoveries were 88%-107% for both assays. The mean difference (95% limits of agreement) between the new total PAPP-A assay and a previously reported total PAPP-A assay was -3.2% (-45.7% to 39.3%; n = 546; P = 0.0019). In serum samples from 159 non-ACS individuals, median concentrations (interquartile range) were 2.42 (1.14) mIU/L for total PAPP-A, 2.20 (1.18) mIU/L for PAPP-A/proMBP, and 0.18 (0.63) mIU/L for noncomplexed PAPP-A. Total PAPP-A and PAPP-A/proMBP, but not noncomplexed PAPP-A, correlated with age (r = 0.290, P = 0.0002; r = 0.230, P = 0.0035; r = 0.075, P = 0.3483, respectively). CONCLUSIONS: The new assays described revealed that noncomplexed PAPP-A is found only in negligible amounts in non-ACS samples.