Total amount of ECP per eosinophil as indicator for the activity state of eosinophils.

Eosinophil cationic protein (ECP) serum concentrations yield additional information regarding the activity of allergic diseases because ECP serum concentrations correlate very well with blood eosinophil counts. We established spontaneous serum ECP release per eosinophil during in vitro coagulation amounting to 0.03-0.5 pg per eosinophil. When comparing the ECP per eosinophil ratio with the eosinophil concentration, a decreasing trend of released ECP in serum was observed towards higher eosinophil counts. The total amount of extractable ECP in eosinophilic granulocytes is determined by means of extraction with application of a 0.5% Cetyl-trimethyl-ammonium-bromide solution. The total amount of extractable ECP in eosinophils amounts to 0.4-17 pg/eosinophil. Results showed a slight tendency to decrease with higher eosinophil counts. Irrespective of their concentration in blood, eosinophils were demonstrated to release 2-4% of their total ECP amount. It is concluded from experiments in this study that release during in vitro clotting or measuring the total amount of extractable ECP does not yield any additional diagnostic value in detecting the degree of activation in eosinophils.

Effect of immunotherapy on eosinophil activation in pollen sensitive children.

Immunotherapy is a well documented method for treatment of children with allergic airway diseases and allergic rhinitis. The aim of this investigation was to establish the effect of immunotherapy on the presence and activation state of eosinophils in pollen sensitive patients, measured by eosinophil counts and their degranulation product in serum, eosinophil cationic protein in comparison with the (specific) IgE concentration in pollen sensitive patients. In both groups intra-individual variations in (specific) IgE concentrations, due to increase of pollen exposure, were not observed. Blood eosinophil counts and serum ECP concentrations showed consistent results after starting immunotherapy. Overall release of ECP per eosinophil remained also unaffected after the start of immunotherapy. Results of ECP concentrations and eosinophil counts in the subjects' group did not show any deviation when compared with the control subjects' group. It is concluded that the laboratory parameters are not helpful in monitoring the efficacy of immunotherapy in patients with seasonal rhinitis.

Surplus value of eosinophil count and ECP to diagnose and monitor asthmatic patients.

BACKGROUND: Eosinophil cationic protein (ESP) is one of the granule proteins in eosinophilic granulocytes. Release of this protein may reflect the activity state of eosinophilic granulocytes in diseased subjects. The purpose of this study was to evaluate the additional value of serum ECP measurement over the eosinophil count, (specific) IgE concentration and CRP concentration in order to evaluate the effects of treatment in patients started on corticosteroids and to distinguish individual patients with asthma from healthy subjects on the basis of laboratory results. METHODS: In a longitudinal study, serial measurements of serum ECP, eosinophil count and other laboratory parameters have been evaluated and compared with spirometry and tests of bronchial hyperresponsiveness in ten asthmatic subjects. The subjects were investigated before therapy was started and 3, 6 and 9 months after the start of therapy with inhaled corticosteroids. Laboratory parameters in the patient group are compared with results obtained from a reference group of apparently healthy subjects (n=223). RESULTS: Statistically significant correlations are observed between blood eosinophil counts and serum ECP concentrations with the hyperresponsiveness tests PC20 (r=0.44 and r=0.46, respectively) and with a decrease in FEV(1) after exercise (r=0.66 and r=0.60, respectively). A significant difference was detected between serum ECP concentrations from the patients' group and from the reference group. However, a wide range of overlapping results was observed between the reference group and the asthmatic patients. CONCLUSIONS: Asthma is a disease which is now more frequently treated by general practitioners. In general practice there is not always the opportunity to evaluate asthma activity by the application of hyperreactivity tests. When hyperreactivity testing is not available, measuring serum ECP concentration or eosinophil blood count would be an alternative method to monitor the effects of corticosteroid treatment. In this study the additional value of serum ECP in addition to the eosinophil count is evaluated to determine the effects of treatment in patients who started with application of corticosteroids. However, because of the analogous correlation coefficients of laboratory parameters with tests regarding hyperresponsiveness, no additional benefit of serum ECP concentration over eosinophil blood count in monitoring the effect of corticosteroids can be detected. The additional value of serum ECP concentrations and eosinophil counts to detect an asthmatic constitution for individual cases is doubtful.

Serum eosinophil cationic protein in active and quiescent ulcerative colitis.

Inflammatory bowel disorders are characterized by an accumulation of eosinophilic granulocytes, mast cells, lymphocytes and neutrophilic granulocytes in the intestinal mucosa. The aim of this study was to examine the concentration of eosinophilic granulocytes in the blood of patients during active ulcerative colitis in comparison with patients during remission and apparently healthy control subjects. Besides counting, the activity grade of eosinophilic granulocytes has been studied by estimation of their degranulation product eosinophil cationic protein. Subjects with active ulcerative colitis could be distinguished from patients with quiescent ulcerative colitis by establishment of the eosinophil cationic protein concentration, neutrophilic granulocyte count, erythrocyte sedimentation rate, C-reactive protein and albumin concentration. After two weeks of corticosteroid treatment, serum eosinophil cationic protein concentrations and eosinophil counts in blood were significantly decreased. A decrease in blood eosinophil count was accompanied by a decrease in eosinophil cationic protein concentrations in serum in most subjects with ulcerative colitis. After twelve weeks of corticosteroid administration, serum albumin concentrations were significantly increased, whereas serum concentrations of C-reactive protein were significantly decreased. During treatment with corticosteroids, serum eosinophil cationic protein concentrations and blood eosinophil counts are appropriate laboratory markers to detect the effect of medication in the course of ulcerative colitis.

Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections.

We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.

Eosinophil cationic protein in serum from nonatopic and asymptomatic atopic individuals after standardized blood clotting at 37 degrees C.

Eosinophil cationic protein (ECP) and ECP/eosinophil ratio were measured in 223 apparently healthy subjects. The serum sample for ECP measurement was obtained by standardized clotting of the blood sample for 2 hours at 37 degrees C. Eosinophil count, ECP concentration and ECP/eosinophil ratio were no different between men (n = 122) and women (n = 101). Reference ranges for serum ECP and ECP/eosinophil ratio were 12-99 micrograms/L and 61-367 micrograms ECP per 10(9) eosinophils, respectively. Serum ECP and blood eosinophil count were positively correlated (y = 141x + 18, R2 = 0.45, P < 0.001). The ECP/eosinophil ratio, however, was found to decrease with increasing eosinophil count. Twenty-three per cent of the apparently healthy subjects were found to have a positive score for immunoglobulin E antibodies specific to inhalant allergens, and thus were considered atopic. Serum ECP concentrations and eosinophil counts were significantly higher in this group compared with the non-atopic group. In this healthy population no decision level for either eosinophil count, serum ECP or ECP/eosinophil ratio could be found that discriminated atopic from non-atopic individuals.

Eosinophil protein X concentration is dependent on eosinophil concentration.

The relationship between the eosinophil concentration and the serum eosinophil protein X concentration was investigated in 80 subjects. Higher eosinophil counts resulted in obviously increased serum eosinophil protein X concentrations. However, the amount of eosinophil protein X released per eosinophil granulocyte is significantly higher in subjects with lower eosinophil counts. Atopic subjects (N = 19) show a significantly higher eosinophil concentration (p = 0.002) and eosinophil protein X concentration (p = 0.004) and a significantly lower eosinophil protein X/eosinophil ratio (p = 0.02), compared with non-atopic subjects (N = 61). However, there appears to be no difference between the concentration of eosinophil protein X in atopic and non-atopic subjects if the eosinophil concentration is taken into account. When using eosinophil protein X as an indicator of eosinophil activation, for instance in asthmatic subjects, the eosinophil count should also be considered for correct clinical interpretation of results.

A Dutch family with Hb Atlanta [beta 75(E19)Leu-->Pro].

We have investigated four members of a three-generation Dutch family for a suspected hemoglobinopathy. Chronic hemolysis and a moderate macrocytic normochromic anemia with slight morphological abnormalities of the red cells was observed in all four. Hemoglobin chain synthesis in vitro and separation of the globin chains by reversed phase high performance liquid chromatography revealed an abnormal beta-globin species in addition to the normal alpha and beta chains. The decreased amount of normal beta-globin and the low amount of unidentified protein suggested an unstable beta-globin variant. An abnormal band was detected by isoelectrofocusing. In one family member tested, the hemoglobin in an erythrocyte lysate had decreased heat stability. All carriers were positive in the isopropanol hemoglobin instability test. Treatment of erythrocytes with methylviolet gave rise to microgranular inclusions. Nucleotide sequencing of the polymerase chain reaction-amplified beta-globin gene revealed a heterozygous single base pair T-->C mutation at codon 75, which changes the normal CTG codon for leucine to a CCG codon for proline. This variant has previously been identified as Hb Atlanta or beta 75(E19)Leu-->Pro. The mutation creates a new Msp I restriction site, which was used to confirm the diagnosis in all four family members. A quantitative reverse transcriptase polymerase chain reaction procedure for determining the relative amounts of mRNA transcripts for the normal and abnormal globin chain showed a comparable stability for both transcripts.

[Molecular-genetic studies of carrier stage of glucose-6-phosphate dehydrogenase deficiency]. / Moleculair-genetisch onderzoek naar draagsterschap van glucose-6-fosfaat-dehydrogenase-deficiëntie.

OBJECTIVE: Determining the reliability of a new DNA analysis in the detection of carriers of 6 mutations that cause glucose-6-phosphate dehydrogenase (G6PD) deficiency. DESIGN: Validation of a diagnostic test. SETTING: Central Laboratory of the Netherlands Red Cross Blood Transfusion Service in Amsterdam, the Netherlands. METHOD: With polymerase chain reactions (PCR) and restriction enzyme analyses, the DNA of 78 proven patients or carriers was compared with the DNA of 51 patients suffering from haemolytic anaemia (possibly due to G6PD deficiency) and of 50 healthy blood donors. RESULTS: In 60 of the 78 patients, 1 or 2 of the 6 mutations were found that lead--according to the literature--to G6PD deficiency. In 2 of the 51 anaemic patients a clinically relevant mutation was found, while such a mutation was revealed in 3 of the 50 blood donors. All 3 had been born in Curaçao or Surinam, areas with a higher incidence of G6PD deficiency than the Netherlands. CONCLUSION: In comparison with G6PD activity tests, which leave 50% of carriers undetected, the described PCR method is a reliable test. Because G6PD activity measurement is independent of mutation analysis, we conclude that a combination of these tests will detect carriers of G6PD deficiency with a higher sensitivity than either of these tests separately.

Effect of clotting temperature and eosinophil concentration on the eosinophil cationic protein concentration in serum.

Earlier investigations have indicated the need for detailed instructions about the incubation conditions of blood samples before establishment of eosinophil cationic protein concentration in serum. Therefore, the effect of different clotting temperatures and eosinophil concentrations on the serum eosinophil cationic protein concentration was quantified in 40 subjects. Our results show that serum eosinophil cationic protein concentrations strongly depend on the clotting temperature. Blood samples clotted for 1 h at 37 degrees C had 5-10 times higher serum eosinophil cationic protein concentrations than blood samples of the same subject clotted for 1 h at 0 degrees C. Higher eosinophil counts resulted also in increased serum eosinophil cationic protein concentrations.