Self-reported food intolerance and mucosal reactivity after rectal food protein challenge in patients with rheumatoid arthritis.

OBJECTIVES: A dietary link to rheumatoid arthritis (RA) has been suspected and an influence on arthritic symptoms by different diets has been reported. Our primary aim was to record the self-experienced adverse food reactions in patients with RA. A secondary aim was to relate self-experienced adverse reactions to dairy produce and wheat to the local mucosal reactivity observed after rectal challenge with cow's milk protein (CM) and wheat gluten. METHODS: A questionnaire about self-experienced adverse reaction to food was sent to 347 RA patients. Rectal challenge with CM and gluten was performed in 27 of these patients and in healthy controls (n = 18). After a 15-h challenge the mucosal production of nitric oxide (NO) and the mucosal release of myeloperoxidase (MPO) and eosinophil cationic protein (ECP) were measured by using the mucosal patch technique. RESULTS: Twenty-seven per cent of the RA patients reported food intolerance (FI) to various foods, and in particular to CM, meat, and wheat gluten. Strong mucosal reactivity to CM was observed in 11% of the patients. Moderately increased mucosal reactivity to CM and gluten was found in 22% and 33%, respectively, of the patients. No relationship was found between self-experienced adverse reactions to CM or gluten and mucosal reactivity to these proteins. CONCLUSIONS: Perceived FI is reported frequently by RA patients, with a prevalence similar to that reported previously in the general population. Mucosal reactivity to CM and gluten is seen in a minor fraction of RA patients and is not related to the frequently perceived intolerance to these proteins.

Gastrointestinal sensitivity to soy and milk proteins in patients with IgA nephropathy.

BACKGROUND: sensitivity to food antigens has been postulated as a contributing factor to the pathogenesis of IgA nephropathy (IgAN). METHODS: in this study we used a recently developed mucosal patch technique to evaluate rectal mucosal sensitivity to soy and cow's milk (CM) proteins in IgAN patients (n = 28) compared to healthy subjects (n = 18). The rectal mucosal production of nitric oxide (NO) and release of myeloperoxidase (MPO) and eosinophil cationic protein (ECP) were measured. Serum samples were analyzed for IgA and IgG antibodies to alpha-lactalbumin, beta-lactoglobulin, casein and soy. RESULTS: 14 of 28 (14/28) patients experienced a rectal mucosal reaction, measured by increased NO and/or MPO levels, upon rectal challenge with soy and/or cow's milk proteins. The levels of IgG antibodies to alpha-lactalbumin, beta-lactoglobulin and casein were significantly higher in CM sensitive as compared with non-sensitive IgAN patients, whereas the mean serum levels of IgA antibodies were similar. No differences were seen in serum levels of IgA or IgG antibodies to soy. CONCLUSION: it is concluded that approximately half of our IgAN patients have a rectal mucosal sensitivity to soy or CM, and that an immune reactivity against antigens may be involved in the pathogenesis of IgAN in this subgroup of patients.

Cardiac troponin I is associated with severity of myxomatous mitral valve disease, age, and C-reactive protein in dogs.

BACKGROUND: Concentrations of cardiac troponin I (cTnI) and C-reactive protein (CRP) might be associated with cardiac remodeling in dogs with myxomatous mitral valve disease (MMVD). Age- and sex-dependent variations in cTnI concentration have been described. OBJECTIVE: To investigate whether plasma concentrations of cTnI and CRP are associated with severity of MMVD, and investigate potential associations of dog characteristics on cTnI and CRP concentrations. ANIMALS: Eighty-one client-owned dogs with MMVD of varying severity. METHODS: Dogs were prospectively recruited for the study. Dogs were classified according to severity of MMVD. Plasma cTnI was analyzed by a high sensitivity cTnI assay with a lower limit of detection of 0.001 ng/mL, and plasma CRP was analyzed by a canine-specific CRP ELISA. RESULTS: Higher cTnI concentrations were detected in dogs with moderate (0.014 [interquartile range 0.008-0.029] ng/mL, P = .0011) and severe (0.043 [0.031-0.087] ng/mL, P < .0001) MMVD, compared with healthy dogs (0.001 [0.001-0.004]ng/mL). Dogs with severe MMVD also had higher cTnI concentrations than dogs with mild (0.003 [0.001-0.024] ng/mL, P < .0001) and moderate (P = .0019) MMVD. There were significant associations of age, CRP, heart rate, and left ventricular end-diastolic diameter, on cTnI concentration C-reactive protein did not differ among severity groups, but was significantly associated with cTnI, breed, and systolic blood pressure on CRP concentration. CONCLUSIONS AND CLINICAL IMPORTANCE: Analysis of cTnI concentration has potential to increase knowledge of overall cardiac remodeling in dogs with MMVD. However, effect of age on cTnI needs consideration when assessing cTnI.

Eosinophil cationic protein stimulates migration of human lung fibroblasts in vitro.

Asthma is characterized by eosinophilic inflammation and remodelling of the airways. Eosinophil cationic protein (ECP) is a protein released by activated eosinophils and the hypothesis that ECP contributes to the development of structural changes in the airways of asthmatics has been posed. Fibroblast recruitment is an important step in the remodelling process, and we therefore put the question whether ECP stimulates migration of human lung fibroblasts. Human peripheral eosinophils isolated from buffycoats from healthy individuals were cultured and conditioned media (CM) were collected. Native ECP was extracted from human peripheral eosinophils by gel filtration, ion-exchange and chelating chromatography. The ability of eosinophil CM and ECP to stimulate fibroblast migration was determined using the 48-well Boyden chamber. ECP concentrations in CM were assayed by ECP-CAP-FEIA. Both CM and ECP significantly stimulated fibroblast migration (48.4+/-cells/field versus 33+/-2 and 36+/-6 versus 25+/-4; P<0.001 and 0.05 respectively) in a time- and concentration-dependent manner. Adding neutralizing ECP antibodies attenuated fibroblast migration induced by both ECP as well as CM. ECP stimulates migration of human lung fibroblasts, suggesting a potential mechanism for eosinophils in the fibrotic response. This may be an important mechanism by which ECP promotes remodelling of extracellular matrix leading to airway fibrosis in asthmatics.

Cow's milk protein sensitivity assessed by the mucosal patch technique is related to irritable bowel syndrome in patients with primary Sjögren's syndrome.

INTRODUCTION: Patients with primary Sjögren's syndrome (pSS) are reported to have a variety of gastrointestinal symptoms partly attributed to an overrepresentation of celiac disease. We have observed that irritable bowel syndrome (IBS)-like symptoms are frequent complaints in this patient group. Allergic manifestations to various drugs are also common in pSS. A role of food allergy in IBS has been proposed. OBJECTIVE: This study is aimed at evaluating the mucosal response to rectal challenge with cow's milk protein (CM) in patients with pSS and relates possible CM reactivity to their intestinal symptoms. METHODS: A rectal challenge with CM was performed in 21 patients with pSS and 18 healthy controls. Fifteen hours after challenge the mucosal production of nitric oxide (NO) and the release of myeloperoxidase (MPO) as signs of mucosal inflammatory reaction were measured using the mucosal patch technique. RESULTS: Eight out of 21 patients with pSS had a definite increase of mucosal NO synthesis and the luminal release of MPO after rectal CM challenge. This sign of milk sensitivity was not linked to IgG/IgA antibodies to milk proteins. The symptoms for IBS according to Rome III criteria were fulfilled in 13 patients. All patients who were CM sensitive suffered from IBS. In a small open study, patients reactive to CM reported an improvement of intestinal symptoms on a CM-free diet. CONCLUSION: A rectal mucosal inflammatory response after CM challenge is seen in 38% of patients with pSS as a sign of CM sensitivity. IBS-like symptoms were common in pSS, linked to CM sensitivity.

Regulation of granulocyte function by hyaluronic acid. In vitro and in vivo effects on phagocytosis, locomotion, and metabolism.

Hyaluronic acid (HA) stimulated the function of polymorphonucler leukocytes (PMN) both in vitro and in vivo. Stimulation in vitro was achieved by the incubation of PMN and HA in heparinized whole blood at concentrations of HA between 5 and 500 microgram/liter. The stimulation of the PMN function was demonstrated by an increase rate of phagocytosis of complement- and/or immunoglobulin (Ig)G-coated latex particles, increased adherence to nylon wool, increased random migration and chemotactic response, increased chemiluminescence during phagocytosis, and raised levels of intracellular ATP. The effect of HA in vivo was demonstrated, after subcutaneous administration of HA (5-20 mg) to healthy volunteers, by an enhanced rate of phagocytosis of the subsequently isolated neutrophils. The duration of the effect of one administration was approximately 1 wk with maximum effect on days 2-4. HA injections to patients with increased susceptibility to bacterial infections and impaired neutrophil function demonstrated an enhanced neutrophil function also in these individuals. HA may therefore be a new principle by which resistance to infections can be enhanced.

Lipocalins as biochemical markers of disease.

Lipocalins as biochemical markers of disease have been used extensively. The clinical indications relate to almost any field of medicine, such as inflammatory disease, cancer, lipid disorders, liver and kidney function. Some of the more well-known lipocalins that have been used as markers of disease are orosomucoid, Protein HC (alpha(1)-microglobulin), apolipoprotein D, retinol-binding protein, complement C8 gamma, prostaglandin D synthase and human tear prealbumin, and these markers will be briefly reviewed in this article. Emphasis, however, will be put on the description of another newly described lipocalin, i.e. human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), since the body fluid measurement of HNL/NGAL was shown to be a superior means to distinguish between acute viral and bacterial infections and also to accurately reflect the activity and involvement of neutrophils in a variety of other diseases.

Troponin T identifies patients with unstable coronary artery disease who benefit from long-term antithrombotic protection. Fragmin in Unstable Coronary Artery Disease (FRISC) Study Group.

OBJECTIVES: We sought to evaluate whether troponin T might be used for identification of patients with unstable coronary artery disease in whom treatment with low molecular weight heparin is beneficial. BACKGROUND: Early identification of subgroups with differences in response to a certain treatment is important to optimize the utilization of different therapeutic approaches. METHODS: Nine-hundred seventy-one patients with unstable coronary artery disease who participated in a trial of the low molecular weight heparin dalteparin (Fragmin) and who provided blood samples were classified into subgroups according to troponin T level. In the short-term phase all patients received subcutaneous dalteparin/placebo twice daily for 6 days. During the long-term phase they continued with daltparin/placebo once daily for another 5 weeks. RESULTS: In the short-term phase, dalteparin reduced the incidence of death or myocardial infarction from 2.4% to 0% (p = 0.12) and from 6.0% to 2.5% (p < 0.05) in 327 and 644 patients with troponin T levels < 0.1 and > or = 0.1 micrograms/liter, respectively. During long-term treatment there was an increasing difference between the placebo and dalteparin group in those with troponin T levels > or = 0.1 microgram/liter, in whom the incidences at 40 days were 14.2% and 7.4%, respectively (p < 0.01). In contrast, no beneficial effect of the long-term treatment could be demonstrated in those with troponin T levels < 0.1 microgram/liter (4.7% vs. 5.7%). CONCLUSIONS: Elevation of troponin T identifies a subgroup of patients in whom prolonged antithrombotic treatment (e.g., with dalteparin) is beneficial.

Mechanisms behind the prognostic value of troponin T in unstable coronary artery disease: a FRISC II substudy.

OBJECTIVES: This study was designed to elucidate possible mechanisms for the prognostic value of troponin T (tnT). BACKGROUND: The reasons for the adverse prognosis associated with elevation of troponins in unstable coronary artery disease are poorly understood. METHODS: Patients enrolled in the Fast Revascularization during InStability in CAD (FRISC-II) trial were included. Clinical characteristics, findings at echocardiography and coronary angiography, and prognosis were evaluated in relation to different tnT levels. RESULTS: Absence of significant coronary stenosis was more frequent and three-vessel disease or left main stem stenosis was less frequent in patients without, compared with, detectable tnT. The occurrence of visible thrombus increased with rising levels of tnT. In the group with the highest levels of tnT, occlusion of the left circumflex artery was more common than in the three other tnT groups, as was a left ventricular ejection fraction below 0.45. The one-year risk of death in the noninvasive arm of the study increased by increasing levels of tnT (1.6% to 4.6%), whereas the risk of myocardial infarction showed an inverted U-shaped curve and was lower in the lowest (5.5%) and highest (8.4%) tnT groups than in the two intermediate groups (17.5% and 16.2%). CONCLUSIONS: Any detectable elevation of tnT raises the probability of significant coronary stenosis and thrombus formation and is associated with an increased risk of reinfarction and death. However, at a more pronounced elevation of troponin, a higher proportion of patients has a persistent occlusion of the culprit vessel and reduced left ventricular function, associated with a high mortality but a modest risk of reinfarction.

Low molecular weight heparin (dalteparin) as adjuvant treatment of thrombolysis in acute myocardial infarction--a pilot study: biochemical markers in acute coronary syndromes (BIOMACS II).

OBJECTIVES: This randomized, double blind, placebo-controlled pilot trial evaluated the effect of dalteparin as an adjuvant to thrombolysis in patients with acute myocardial infarction regarding early reperfusion, recurrent ischemia and patency at 24 h. BACKGROUND: Low-molecular-weight heparin, given subcutaneously twice daily without monitoring, might be an attractive alternative to conventional intravenous heparin in the treatment of acute myocardial infarction. METHODS: In 101 patients dalteparin/placebo 100 IU/kg was given just before streptokinase and a second injection 120 IU/kg after 12 h. Monitoring with continuous vector-ECG was done to obtain signs of early reperfusion and later ischemic episodes. Blood samples for myoglobin were obtained at start and after 90 min to evaluate signs of reperfusion. Coronary angiography was performed after 20-28 h to evaluate TIMI-flow in the infarct-related artery. RESULTS: Dalteparin added to streptokinase tended to provide a higher rate of TIMI grade 3 flow in infarct-related artery compared to placebo, 68% versus 51% (p = 0.10). Dalteparin had no effects on noninvasive signs of early reperfusion. In patients with signs of early reperfusion, there seemed to be a higher rate of TIMI grade 3 flow, 74% versus 46% (myoglobin) (p = 0.04) and 73% versus 52% (vector-ECG) (p = 0.11). Ischemic episodes 6-24 h. after start of treatment were fewer in the dalteparin group, 16% versus 38% (p = 0.04). CONCLUSIONS: When dalteparin was added as an adjuvant to streptokinase and aspirin, there were tendencies for less ECG monitoring evidence of recurrent ischemia and better patency at 24 h, warranting further study.

Bronchial mucosal mast cells in asymptomatic smokers relation to structure, lung function and emphysema.

The pathologic mechanisms of chronic obstructive pulmonary disease (COPD) most certainly involves neutrophil granulocytes, cytotoxic T-cells, macophages and mast cells. The aim of this study was to investigate the relation between the number of mast cells in different compartments in bronchial biopsies of central proximal airways to structural changes, lung function tests and emphysema detected by high resolution computed tomography (HRCT). Twenty nine asymptomatic smoking and 16 never-smoking men from a population study were recruited. Central bronchial biopsies were stained to identify mast cells by immunohistochemistry. The number of mast cells in the epithelium, lamina propria and smooth muscle as well as epithelial integrity and thickness of the tenascin and laminin layer were determined. Smokers had increased numbers of mast cells in all compartments (P<0.001). Structural changes were correlated to mast cell numbers with the closest associations to mast cell numbers in the smooth muscle [epithelial integrity (R(S)=-0.48, P=0.008), laminin layer (R(S)=0.63, P=0.0002), tenascin layer (R(S)=0.40, P=0.03)]. Similar correlations between mast cells and lung function tests were seen [functional residual capacity (FRC) (R(S)=0.60, P=0.0006), total lung capacity (TLC) (R(S)=0.44, P=0.02) and residual volume (RV) (R(S)=0.41, P=0.03)]. No correlations could be detected between mast cells and FEV1 or to emphysema. Smoking is associated with an increase of mast cells in all compartments of the bronchial mucosa, including smooth muscle, and this is related to altered airway structure and function.

Ultrastructure of bronchial biopsies from patients with allergic and non-allergic asthma.

Epithelial damage is commonly found in airways of asthma patients. The aim of this study was to investigate epithelial damage in allergic and non-allergic asthma at the ultrastructural level. Bronchial biopsies obtained from patients with allergic asthma (n=11), non-allergic asthma (n=7), and healthy controls (n=5) were studied by transmission electron microscopy. Epithelial damage was found to be extensive in both asthma groups. Both in basal and in columnar cells, relative desmosome length was reduced by 30-40%. In columnar cells, half-desmosomes (i.e., desmosomes of which only one side was present) were frequently noticed. Eosinophils showing piece-meal degranulation were commonly observed in allergic asthma. Degranulating mast cells were more often observed in allergic asthma. Goblet cell hyperplasia was only found in allergic asthma. Lymphocytes were increased in both groups. In both groups, the lamina densa of the basal lamina was thicker than the control by about 40-50%. In allergic asthma the lamina densa was irregular with focal thickening. While there was always a tendency for changes (epithelial damage, desmosomes, degranulating mast cells, basal lamina) to be more extensive in allergic asthma compared to non-allergic asthma, there was no significant difference between the two groups in this respect. Reduced desmosomal contact may be an important factor in the epithelial shedding observed in patients with asthma.

The chemotactic response of granulocytes to the low molecular weight chemoattractants f-MLP, C5f, and LTB4 is dependent on chemokinetic factors.

The dependence of the low molecular weight chemoattractants (LMCs) formylmethionyl-leucylphenylalanine (f-MLP), C5f, and leukotriene B4 (LTB4) on albumin to express their chemotactic activity towards granulocytes (PMNs) was investigated in order to study the required qualities of albumin and if albumin could be replaced by any other proteins. The results demonstrated that the supporting effect of isolated albumin was dependent on the method of purification. Only isolated albumin exposed to ethanol precipitation during the purification procedure supported the chemotactic effect of LMCs. The albumin preparation that supported the effect of LMCs also mediated a chemokinetic effect on PMN migration. Albumin isolated by methods other than ethanol precipitation neither exerted a chemokinetic effect nor supported the chemotactic effect of LMCs. Heated, normal serum and isolated alpha 1-antitrypsin supported the chemotactic activity of LMCs and also mediated a chemokinetic effect on PMN migration. The present investigation suggests an important role for the chemokinetic factors, since it is indicated that their presence is necessary for the chemotactic response of PMNs to the low molecular weight chemoattractants C5f; LTB4, and f-MLP.

Inhibition of neutrophil and eosinophil chemotactic responses to PAF by the PAF-antagonists WEB-2086, L-652,731, and SRI-63441.

The influence of the three PAF-antagonists WEB-2086, L-652,731, and SRI-63441 on the chemotactic response of neutrophil and eosinophil granulocytes to PAF was investigated. When the PAF-antagonists were added to the cell suspension that was exposed to a gradient of PAF, WEB-2086 and SRI-63441 at the concentration of 10(-6) mol/litre inhibited (P less than .01) the neutrophil and eosinophil chemotactic response to 10(-8) and 10(-9) mol PAF per litre; at the concentration of 5 x 10(-6) mol/litre, WEB-2086 and SRI-63441 also inhibited (P less than .02) the response to 10(-7) mol PAF per litre. Under the same conditions L-652,731 at the concentration of 5 x 10(-6) mol/litre inhibited (P less than .01) the eosinophil chemotactic response to 10(-8) and 10(-9) mol PAF per litre. The inhibition of the chemotactic response to PAF by the three PAF-antagonists was specific, since the chemotactic response to C5f, f-MLP, and LTB4 was not affected by WEB-2086, L-652,731, or SRI-63441, neither was the chemokinetic migration induced by albumin.

New method for the measurement of eosinophil migration.

A new application of the Boyden chamber method for the measurement of eosinophil migration, without the need of eosinophil isolation, has been developed. A cell suspension containing a mixture of granulocytes, neutrophils to the greater part, was used. The eosinophils were identified by staining their granules with Chromotrope 2R. The method made it possible to study the migration of eosinophils from normal individuals without eosinophilia. Control experiments demonstrated that the chemotactic and chemokinetic response of eosinophils in the granulocyte mixture was in accordance with the response of isolated eosinophils from the same donor. Normal eosinophils demonstrated a significant chemotactic response to C5f, platelet-activating factor (PAF), leukotriene B4 (LTB4), and f-meth-leu-phe (f-MLP). Furthermore, PAF was demonstrated to be significantly more eosinophil chemotactic than neutrophil chemotactic.

Urinary neutrophil gelatinase-associated lipocalin to hepcidin ratio as a biomarker of acute kidney injury in intensive care unit patients.

BACKGROUND: Labile iron is important in the pathogenesis of acute kidney injury (AKI). Neutrophil gelatinase-associated lipocalin (NGAL) and hepcidin control iron metabolism and are upregulated during renal stress. However, higher levels of urinary NGAL are associated with AKI severity whereas higher urinary hepcidin levels are associated with absence of AKI. We aimed to investigate the value of combining both biomarkers to estimate the severity and progression of AKI in intensive care unit (ICU) patients. METHODS: Urinary NGAL and hepcidin were quantified within 48 hours of ICU admission in patients with the systemic inflammatory response syndrome and early kidney dysfunction (oliguria for ≥ 2 hours and/or a 25 µmol/L creatinine rise from baseline). Diagnostic and prognostic characteristics were assessed by logistic regression and receiver operating characteristics (ROC) analysis. RESULTS: Of 102 patients, 26 had mild AKI and 28 patients had severe AKI on admission. Sepsis (21%), cardiac surgery (17%) and liver failure (9%) were primary admission diagnoses. NGAL increased (P=0.03) whereas hepcidin decreased (P=0.01) with increasing AKI severity. The value of NGAL/hepcidin ratio to detect severe AKI was higher than when NGAL and hepcidin were used individually and persisted after adjusting for potential confounders (adjusted OR 2.40, 95% CI 1.20-4.78). The ROC areas for predicting worsening AKI were 0.50, 0.52 and 0.48 for NGAL, 1/hepcidin and the NGAL/hepcidin ratio. CONCLUSION: The NGAL/hepcidin ratio is more strongly associated with severe AKI than the single biomarkers alone. NGAL and hepcidin, alone or combined as a ratio, were unable to predict progressive AKI in this selected ICU cohort.

Eosinophil cationic protein alters proteoglycan metabolism in human lung fibroblast cultures.

Eosinophil cationic protein (ECP), a highly basic protein secreted from eosinophilic granulocytes, has been shown to take part in the inflammatory reaction. The involvement of ECP in fibroblast activation was therefore investigated in cell culture. Production of proteoglycans, hyaluronan and collagen in the presence of ECP was measured after incorporation of radioactive precursors and separation into different proteoglycan classes using gel and ion exchange chromatography and hydrophobic interaction chromatography. Proteoglycan accumulation in the cell layer was increased two- to fivefold at an ECP-concentration of 10 micrograms/ml. No effect on collagen, other proteins or hyaluronan was noted. Furthermore, no effect was observed on cell proliferation. The increased proteoglycan accumulation could be inhibited by addition of heparin or of antibodies to ECP. The effect could not be mimicked by the two basic peptides protamine and poly-L-lysine, speaking in favor of specificity. The increase in proteoglycan material was seen exclusively in the intracellular pool. No change of proteoglycans in the medium or the cell surface-associated pool was noted. The increase in the cell layer was accounted for by a two- to fivefold increase in free chains of heparan sulfate and dermatan sulfate. No change was seen in the proteoglycan pattern. No effect on proteoglycan synthesis or on endocytosis was noted. The increased accumulation of polysaccharide was caused by inhibited degradation of glycosaminoglycans. The half-lives of large and small heparan sulfate proteoglycans/glycosaminoglycans and dermatan sulfate proteoglycans/glycosaminoglycans in the cell layer are increased four- to sevenfold. We conclude that ECP inhibits proteoglycan degradation in fibroblasts, which indicates a role for the eosinophil in generation of fibrosis.

Eosinophil cationic protein (ECP).

ECP (eosinophil cationic protein) was first purified from human myleoid cells in 1971 and identified as an eosinophil granule protein in 1975. ECP is a heterogeneous protein with molecular weights of the variants from 16-24 kDa. ECP is extremely basic with a pI of pH 10.8. The gene for ECP is found on chromosome 14 adjacent to other proteins of the ribonuclease family, with which ECP shares some sequence homologies. ECP has a variety of biological activities interacting with other immune cells and plasma proteins such as coagulation factors and proteins of the complement system. The cytotoxic activity, however, is the most conspicuous. The different isoforms of ECP seem to have different biological properties with respect to cytotoxicity and the effects on fibroblasts. ECP can be measured in biological fluids, by means of sensitive immunoassays, as an indication of eosinophil turnover and activity in vivo.

A method to study apoptosis in eosinophils by flow cytometry.

The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FACS-Scan cell sorter and by Annexin V-coated magnetic beads to permit separate analyses of PI-staining pattern, DNA electrophoresis, and light microscopic examination of the cells in population B. The present study suggest that it is possible to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.

Detection of eosinophil cationic protein (ECP) by an enzyme-linked immunosorbent assay.

Eosinophil cationic protein (ECP) is a highly basic and potent cytotoxic single-chain zinc-containing protein present in the granules of the eosinophilic granulocytes. ECP appears to be involved in defence against parasites and in the tissue damage seen in subjects with allergic and inflammatory disease. To investigate ECP release from in vitro activated human eosinophils and to study the involvement of eosinophils in health and disease, we have developed a sensitive and specific enzyme immunoassay. ECP was purified from normal human peripheral blood eosinophils and polyclonal antibodies to ECP were subsequently raised in rabbits. The ELISA utilizes the biotin/avidin method and measures ECP within the range 15-1000 ng/l. The intra- and interassay coefficients of variation were 6% and 10%, respectively, and the recoveries of 12 and 25 pg of purified ECP added to diluted serum samples were 108 +/- 14.5% (mean +/- SD, n = 12) and 107 +/- 7.5%, respectively. The high sensitivity, reproducibility and specificity of this ELISA makes it suitable for the determination of minute amounts of ECP in in vitro systems as well as in various biological fluids.