Sputum basophils are increased in eosinophilic asthma compared with non-eosinophilic asthma phenotypes.

Sputum basophil numbers are increased in allergic asthmatics, but it is unclear what role airway basophils play in "TH2-low" asthma phenotypes. Using flow cytometry, we found that basophils were significantly increased in all asthmatics (n=26) compared with healthy controls (n=8) (P=0.007) with highest levels observed in eosinophilic asthma (EA); median 0.22%, IQR 0.11%-0.47%; n=14) compared with non-EA (NEA) (0.06%, 0.00%-0.20%; n=12; P<0.05). In asthmatics, basophils were positively correlated with sputum eosinophils (r=0.54; P<0.005) and inversely with sputum neutrophils (r=-0.46: P<0.05), but not with FEV1 (% predicted), FEV1 /FVC or bronchodilator reversibility. In a subgroup initially identified as inadequately controlled asthma (n=7), there was a trend (P=0.08) towards a reduction in sputum basophils following increased inhaled corticosteroid (ICS) treatment. Our findings suggest that basophils may be particularly important in eosinophilic asthma and that sputum basophil assessment could be a useful additional indicator of "TH2-high" asthma.

Asthma control in a random sample of New Zealand adolescent asthmatics.

Asthma control, defined by asthma symptoms and lung function, and asthma medication use, was assessed in 123 adolescent asthmatics. Sputum eosinophilia (>or= 2.5% eosinophils) and bronchial hyperresponsiveness (BHR) to hypertonic saline were also measured to assess whether these additional objective parameters might aid in determining asthma control; 54.5% of subjects had adequately controlled asthma; 50.4% of all subjects reported inhaled corticosteroid use in the preceding 12 months; however, only 22.3% reported regular use. Although BHR and median eosinophil numbers were significantly higher in the inadequately controlled asthmatics, BHR and sputum eosinophilia had poor sensitivity for detecting inadequate asthma control.

Substrates and products of eosinophil peroxidase.

Eosinophil peroxidase has been implicated in promoting oxidative tissue damage in a variety of inflammatory conditions, including asthma. It uses H(2)O(2) to oxidize chloride, bromide and thiocyanate to their respective hypohalous acids. The aim of this study was to establish which oxidants eosinophil peroxidase produces under physiological conditions. By measuring rates of H(2)O(2) utilization by the enzyme at neutral pH, we determined the catalytic rate constants for bromide and thiocyanate as 248 and 223 s(-1) and the Michaelis constants as 0.5 and 0.15 mM respectively. On the basis of these values thiocyanate is preferred 2.8-fold over bromide as a substrate for eosinophil peroxidase. Eosinophil peroxidase catalysed substantive oxidation of chloride only below pH 6.5. We found that when eosinophil peroxidase or myeloperoxidase oxidized thiocyanate, another product besides hypothiocyanite was formed; it also converted methionine into methionine sulphoxide. During the oxidation of thiocyanate, the peroxidases were present as their compound II forms. Compound II did not form when GSH was included to scavenge hypothiocyanite. We propose that the unidentified oxidant was derived from a radical species produced by the one-electron oxidation of hypothiocyanite. We conclude that at plasma concentrations of bromide (20-120 microM) and thiocyanate (20-100 microM), hypobromous acid and oxidation products of thiocyanate are produced by eosinophil peroxidase. Hypochlorous acid is likely to be produced only when substrates preferred over chloride are depleted. Thiocyanate should be considered to augment peroxidase-mediated toxicity because these enzymes can convert relatively benign hypothiocyanite into a stronger oxidant.

Nitrite as a substrate and inhibitor of myeloperoxidase. Implications for nitration and hypochlorous acid production at sites of inflammation.

Myeloperoxidase is a heme enzyme of neutrophils that uses hydrogen peroxide to oxidize chloride to hypochlorous acid. Recently, it has been shown to catalyze nitration of tyrosine. In this study we have investigated the mechanism by which it oxidizes nitrite and promotes nitration of tyrosyl residues. Nitrite was found to be a poor substrate for myeloperoxidase but an excellent inhibitor of its chlorination activity. Nitrite slowed chlorination by univalently reducing the enzyme to an inactive form and as a consequence was oxidized to nitrogen dioxide. In the presence of physiological concentrations of nitrite and chloride, myeloperoxidase catalyzed little nitration of tyrosyl residues in a heptapeptide. However, the efficiency of nitration was enhanced at least 4-fold by free tyrosine. Our data are consistent with a mechanism in which myeloperoxidase oxidizes free tyrosine to tyrosyl radicals that exchange with tyrosyl residues in peptides. These peptide radicals then couple with nitrogen dioxide to form 3-nitrotyrosyl residues. With neutrophils, myeloperoxidase-dependent nitration required a high concentration of nitrite (1 mM), was doubled by tyrosine, and increased 4-fold by superoxide dismutase. Superoxide is likely to inhibit nitration by reacting with nitrogen dioxide and/or tyrosyl radicals. We propose that at sites of inflammation myeloperoxidase will nitrate proteins, even though nitrite is a poor substrate, because the co-substrate tyrosine will be available to facilitate the reaction. Also, production of 3-nitrotyrosine will be most favorable when the concentration of superoxide is low.

Thiocyanate and chloride as competing substrates for myeloperoxidase.

The neutrophil enzyme myeloperoxidase uses H2O2 to oxidize chloride, bromide, iodide and thiocyanate to their respective hypohalous acids. Chloride is considered to be the physiological substrate. However, a detailed kinetic study of its substrate preference has not been undertaken. Our aim was to establish whether myeloperoxidase oxidizes thiocyanate in the presence of chloride at physiological concentrations of these substrates. We determined this by measuring the rate of H2O2 loss in reactions catalysed by the enzyme at various concentrations of each substrate. The relative specificity constants for chloride, bromide and thiocyanate were 1:60:730 respectively, indicating that thiocyanate is by far the most favoured substrate for myeloperoxidase. In the presence of 100 mM chloride, myeloperoxidase catalysed the production of hypothiocyanite at concentrations of thiocyanate as low as 25 microM. With 100 microM thiocyanate, about 50% of the H2O2 present was converted into hypothiocyanite, and the rate of hypohalous acid production equalled the sum of the individual rates obtained when each of these anions was present alone. The rate of H2O2 loss catalysed by myeloperoxidase in the presence of 100 mM chloride doubled when 100 microM thiocyanate was added, and was maximal with 1mM thiocyanate. This indicates that at plasma concentrations of thiocyanate and chloride, myeloperoxidase is far from saturated. We conclude that thiocyanate is a major physiological substrate of myeloperoxidase, regardless of where the enzyme acts. As a consequence, more consideration should be given to the oxidation products of thiocyanate and to the role they play in host defence and inflammation.

Molecular characterization of antigenic polymorphisms (Ond(a) and Mart(a)) of the beta 2 family recognized by human leukocyte alloantisera.

We show that the previously described alloantisera Ond and Mart, which recognize the alloantigens Ond(a) and Mart(a), react with polymorphic variants of alpha L and alpha M subunits of the beta 2 integrin family (CD11a and CD11b molecules). This was shown by testing the alloantisera in a monoclonal antibody-specific immobilization of leukocyte antigens, immunoprecipitation, and immunofluorescence assay against cells from normal donors and from patients with leukocyte adhesion deficiency (beta 2 intergrin deficient). To elucidate the molecular basis of the Ond(a) and Mart(a) alloantigens, RNA was isolated from mononuclear leukocytes derived from individuals of known serologic phenotype. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to amplify the entire coding region of the alpha L and alpha M mRNAs. The Ond(a) antigen was found to be due to a G2466C substitution in the DNA coding for the alpha L subunit, which predicts an Arg766Thr amino-acid polymorphism. The Mart(a) antigen was also found to be due to a single nucleotide substitution (G302A) in the DNA coding for the alpha M subunit, which predicts an Arg61His amino acid polymorphism. Using allele-specific restriction enzyme analysis, the association between point mutations and phenotypes was confirmed. The localization of these alloantigens on integrin molecules further illustrates the polymorphic nature of this class of proteins. Whether the polymorphisms influence the adhesive capacity of the leukocyte integrins remains to be investigated.

Further characterization of the NB 1 antigen as a variably expressed 56-62 kD GPI-linked glycoprotein of plasma membranes and specific granules of neutrophils.

The human neutrophil-specific alloantigen NB1 was identified as a glycosyl-phosphatidylinositol (GPI)-anchored N-glycosylated protein of M(r) 56-62 kD under reducing conditions. Under non-reducing conditions its M(r) was 49-55 kD. This glycoprotein antigen was found to be expressed by only a subpopulation of normal donor neutrophils, and could not be detected on other blood cells. The allotypic epitope recognized by human anti-NB1 IgG was also recognized by the mouse monoclonal antibody 1B5. The percentage of neutrophils stained by these antibodies varied greatly among healthy donors (range 0-100%). When 16 donors were repeatedly tested, the NB1-positive neutrophil fraction appeared to remain remarkably constant over time in most donors, but significant fluctuations were seen in some. NB1 antigen was found to be expressed not only on the plasma membrane, but also intracellularly on the membranes of small vesicles and specific granules. The neutrophils which expressed NB1 antigen on the plasma membrane were the same as those with intracellular expression of this antigen. Crosslinking of NB1 antigen on the plasma membrane with monoclonal antibody 1B5 and goat-anti-mouse Ig resulted in internalization of the complex, while in-vitro stimulation of neutrophils caused an increase of the intensity of plasma membrane staining with anti-NB1, but only of those cells that were positive already prior to stimulation. The NB1 glycoprotein thus appears to identify a distinct subset of neutrophils, the size of which greatly varies among healthy donors. The function of the NB1 glycoprotein remains unclear, but its behaviour upon crosslinking and chemotactic peptide stimulation suggests a possible role as receptor molecule.

Idiopathic thrombocytopenic purpura in pregnancy: a randomized trial on the effect of antenatal low dose corticosteroids on neonatal platelet count.

OBJECTIVE: To determine the efficacy of antenatal low dose oral betamethasone in preventing neonatal thrombocytopenia and/or bleeding in infants of mothers with idiopathic thrombocytopenic purpura (ITP). SETTING: Hospital department of obstetrics and gynaecology, referral centre. PATIENTS: 41 pregnancies in 38 women were randomized. The results of 13 pregnancies were considered non-assessable. The final analysis involved 14 in the betamethasone group and 14 in the non-treatment group. All fulfilled the criteria for ITP. INTERVENTIONS: The treated group received 1.5 mg betamethasone orally per day, from day 259 till day 273 and 1 mg from day 273 till delivery. MAIN OUTCOME MEASURES: Effects of treatment were assessed in terms of maternal platelet counts after the first trimester and neonatal platelet counts at birth and the first week of life and neonatal bleeding episodes. RESULTS: There were no significant differences in neonatal platelet counts at birth. Two infants in the betamethasone group and one in the untreated group had a severe thrombocytopenia either at birth or during the first week of life (less than 50 x 10(9)/l). Seven infants in the betamethasone group and six in the non-treatment group had a mild thrombocytopenia. The overall frequency of neonatal thrombocytopenia was similar: 64% in the betamethasone group and 57% in the untreated group (95% CI of the true difference: -43.5% to +29.5%). There was also no significant difference in neonatal bleeding episodes. CONCLUSIONS: Low-dose betamethasone in pregnant women with ITP does not prevent thrombocytopenia or bleeding in their newborn infants.

Assignment of the gene(s) involved in the expression of the proliferation-related Ki-67 antigen to human chromosome 10.

The antigen recognized by the monoclonal antibody Ki-67 is a proliferation-related nucleolus-associated constituent used as a marker for cycling cells in tumor diagnosis. Antibody Ki-67 reacts with human proliferating cells, but not with hamster and mouse cells. Expression of the Ki-67 antigen was studied in a panel of human-rodent somatic cell hybrids. The results indicate that a gene involved in the expression of the antigen is located on chromosome 10.

Autoimmunity against blood cells in human immunodeficiency-virus (HIV) infection.

In persons with AIDS or at risk from AIDS, autoantibodies against platelets and granulocytes were frequently detected. Platelet-bound immunoglobulins were demonstrated by immunofluorescence in all 16 patients with AIDS, in five out of seven patients with AIDS-related complex/persistent generalized lymphadenopathy (ARC/PGL) and even in seven of 10 healthy sexually active homosexual men. Granulocyte-bound immunoglobulins were found by immunofluorescence in 12 of the 16 AIDS patients, five of the seven patients with ARC/PGL and two of the 10 symptomless men. Red cell bound immunoglobulins were not detected. All patients with AIDS and ARC/PGL and three of the symptomless men were seropositive for human immunodeficiency virus (HIV). The platelet- and granulocyte-bound immunoglobulins could be eluted in 93% and 67% of the cases, respectively. This indicates that specific autoantibodies, rather than circulating immune complexes, which were frequently increased, accounted for the findings. There was no relation between the serological findings and the platelet and granulocyte counts. We conclude that autoantibodies against platelets and granulocytes are common in patients with AIDS and those at risk.

Clinical significance of positive platelet immunofluorescence test in thrombocytopenia.

The sensitivity and specificity of the platelet immunofluorescence test for the diagnosis of idiopathic thrombocytopenia (ITP) was studied in a series of 255 patients. Patients' platelets were tested directly. Diethyl-ether eluates of these platelets and patients' sera were tested indirectly with normal donor platelets. When all three tests were considered, positive results were obtained for 92.0% of the ITP patients with a platelet count of less than 150 X 10(9)/l and for 98.4% of the patients with a count of less than 100 X 10(9)/l. However, for many patients rather weak test results were obtained, with a score of 1/2-1 in 59.8% of the patients. Most patients (94.1%) with a positive direct test had a positive indirect test on the eluate. Thus, platelet-bound antibodies but not platelet-bound immune complexes were present in most, if not all, patients. Positive immunofluorescence tests were obtained for many patients with a diagnosis other than ITP. This resulted in a low specificity of the test for the diagnosis of ITP, evidently because autoimmune thrombocytopenia occurred together with many other diseases and also because antibodies against platelet cryptantigens (expressed by the action of EDTA or by platelet fixation) were present in many patients.

Alloimmunization against the platelet-specific Zwa antigen, resulting in neonatal alloimmune thrombocytopenia or posttransfusion purpura, is associated with the supertypic DRw52 antigen including DR3 and DRw6.

The strong association between HLA-DR3 and Zwa alloimmunization in Zwa-negative mothers, resulting in the production of anti-Zwa antibodies and ultimately in neonatal alloimmune thrombocytopenic purpura (NAITP) in the newborn, could be confirmed. In addition, we have shown an equally strong association between HLA-DR3 and Zwa alloimmunization in Zwa-negative recipients of blood transfusions, resulting in posttransfusion purpura (PTP). However, the strongest association both in mothers of NAITP patients and in PTP patients was observed with the supertypic DRw52 antigen. In fact, all individuals carried this antigen. Normally, DRw52 includes DR3, DR5, DRw6, and DRw8 almost completely. However, in our material, DRw52 included only DR3 and DRw6 with one exception. Another interesting observation is that all PTP patients were women with previous pregnancies without any clinical signs of a bleeding disorder in their children. This indicates that alloimmunization against Zwa during pregnancy is DR3- and/or DRw52-positive women does not always lead to the development of NAITP, but these women may be at risk for the development of PTP after blood transfusion.

Thrombocytopenia associated with gold therapy: a drug-induced autoimmune disease?

We studied 13 patients with rheumatoid arthritis (RA) and gold-induced thrombocytopenia. Platelet-specific autoantibodies of the IgG and often also of the IgM class were detected by immunofluorescence on the patient's platelets and in ether eluates from these platelets. In nine patients we also detected autoantibodies in the serum. The antibodies were unreactive with platelets from patients with Glanzmann's disease in most cases, and were not EDTA dependent. Thus, they had the serological characteristics of true autoantibodies. The reaction of the antibodies with platelets was not influenced by the addition of extra gold. By atomic absorption spectrophotometry we found that the ether eluates, which were often strongly reactive with donor platelets, were free of gold. This indicated that the autoantibodies in the thrombocytopenic patients were not dependent on gold for their reaction. The possibility is raised that gold treatment of rheumatoid arthritis patients induces or enhances the formation of platelet-specific autoantibodies, and that gold-induced thrombocytopenia is a drug-induced autoimmune disease.

The serology of febrile transfusion reactions.

Sera from 40 patients with febrile, nonhemolytic transfusion reactions were tested for the presence of alloantibodies using a number of techniques, including immuno-fluorescence tests on granulocytes, lymphocytes and platelets, a modified NIH lymphocytotoxicity test and the leukocyte agglutination test. Cells of at least 9 donors were used as target cells. Alloantibodies were detected in all sera. The frequency of the occurrence of antibodies was not much higher in sera obtained about 1 month after the transfusion reaction as in sera obtained within 4 days. Most of these antibodies were anti-HLA, but quite frequently platelet-specific antibodies were found, and sometimes these were the only antibodies detected. Granulocyte-specific antibodies were the least frequent. The nature of the antibodies was specified by their difference in reactivity with the cells of multiple donors, by applying panels of cells from typed donors and by absorption and elution experiments. It appeared that not only granulocyte-specific but also HLA- and perhaps platelet-specific antibodies may be responsible for a febrile transfusion reaction. We did not find that the occurrence of rigors, together with fever, was associated with particular serologic results.

Platelet autoantibodies in septicaemia.

Thirty-three patients, 16 of whom had a platelet count below 100 X 10(9)/l, were studied for the presence of platelet antibodies using the platelet suspension immunofluorescence test (PSIFT). In the acute phase platelet-bound immunoglobulins (PBIg) were found in 62% of the thrombocytopenic patients, but also in 65% of the patients with a normal platelet count. Fourteen patients were re-examined after successful treatment of their infection. At that time all had a normal platelet count, yet PBIg were found in 71%. In all but four patients the PBIg could be eluted with ether and were shown to bind to normal platelets but not to Glanzmann type I platelets in 11 of 12 patients tested, indicating the platelet-specific autoantibody nature of the PBIg. Platelet autoantibodies were also demonstrated in the sera: in 38% in the acute phase and in 43% after treatment. In those patients in whom this could be tested the antibodies appeared to be strictly or partially EDTA-dependent. It is postulated that the antibodies are mainly directed against cryptantigens, exposed in vivo only on platelets damaged by the septicaemia and in vitro in the presence of EDTA. This would also explain the lack of relationship between the platelet count and the serological findings.

Red cell, white cell and platelet autoantibodies in visceral leishmaniasis.

The blood of twelve patients with parasitologically proved visceral leishmaniasis was examined for the presence of antibodies against blood cells. The direct antiglobulin test (Coombs' test) was positive in ten. IgG and complement were found on the red cells (alone or combined), but no IgA or IgM. In the serum of two, warm antibodies were found in the bromelin test. Pseudo-agglutination was detected in 8. Investigations for white cell antibodies included the lymphocytotoxicity test (positive in 10), the leuco-agglutination test (positive in 1) and the indirect immunofluorescence test on lymphocytes (none positive) and granulocytes (2 positive). Platelet autoantibodies were demonstrated in the direct (10 positive) and indirect (7 positive) platelet suspension immunofluorescence test. How important these various antibodies are in influencing the lifespan and functions of the respective cells and thus in the pathogenesis of the pancytopenia of kala-azar, can not be concluded from this work but the absence of a direct correlation between haematological values and presence or absence of antibodies and/or their titres argues against a primary role of these antibodies.