Lack of effect of the 5-lipoxygenase inhibitor zileuton in blocking oral aspirin challenges in aspirin-sensitive asthmatics.

BACKGROUND: Leukotrienes (LTs) have been implicated as major mediators of aspirin-(ASA)-induced respiratory reactions. It was therefore logical to assume that an inhibitor of 5-lipoxygenase (5-LO), such as zileuton, given before and during oral challenges with ASA, might prevent ASA-induced respiratory reactions. Indeed, in prior studies, pretreatment of ASA-sensitive respiratory disease patients with leukotriene modifiers eliminated or attenuated respiratory reactions upon re-challenge with the previously established provoking dose of ASA. However, doses higher than the provoking doses were not administered during these reported studies. OBJECTIVE: We wished to determine whether zileuton pretreatment could prevent ASA-induced respiratory reactions in our six volunteers with aspirin-sensitive respiratory disease when ASA challenge doses were started below the usual provoking dose of 60 mg and then increased until a respiratory reaction occurred. METHOD: Aspirin sensitivity was established previously in all six patients during a prior ASA oral challenge. In this study, pretreatment with zileuton 600 mg qid was initiated 7 days prior to, and continued during oral ASA challenges. Patients underwent single-blind oral ASA challenges with escalating doses of ASA, every 3 hours, according to our standard protocol. RESULTS: All six patients reacted to doses of ASA between 45 and 325 mg. Four patients experienced bronchospasm (FEV1 declined 19% to 53%) while receiving zileuton. All six had naso-ocular reactions. Concentrations of urine LTE4 also increased significantly (mean 334 pg/mg Cr at baseline, increasing to 1024 pg/mg Cr at respiratory reactions). CONCLUSIONS: During ASA challenges, zileuton, in standard doses of 600 mg qid was associated with increased synthesis of LTs in five of six patients and naso-ocular reactions in all six patients, as well as bronchospasm in four patients.

Mastocytosis: diverse presentations and outcomes.

Within the general category of mastocytosis lies an array of clinical presentations with differing prognostic implications. We report 3 cases of systemic mastocytosis distinguished by novel aspects of the disease. Case 1 documents the first successful orthotopic liver transplantation in a patient with mastocytosis; case 2 depicts a potential hereditary component of mastocytosis; and case 3 documents the progression of mastocytosis with hematologic abnormality to mast cell leukemia. Future investigations, such as the early definition of c-kit receptor mutations, may provide additional insight as to the molecular basis for this heterogeneous disease and guidance for prognostic implications and targeted therapies.

Identification of a subset of patients with scleroderma with severe pulmonary and vascular disease by the presence of autoantibodies to centromere and histone.

OBJECTIVES: The role of autoantibodies in the investigation and management of rheumatic diseases is well recognised. The objective of this study was to determine the clinical significance of the co-occurrence of antibodies to centromere and histone in serum samples from patients investigated for systemic rheumatic diseases. METHODS: Serum samples from 1316 consecutive patients were screened for antinuclear antibodies and the clinical findings in patients with antibodies to centromere alone were compared with those with antibodies to both centromere and histone. RESULTS: Twenty six patients had antibodies to centromere. Fourteen patients had antibodies to centromere alone and 12 patients had antibodies to centromere and histone. Four of the 12 patients with antibodies to centromere and histone had diffuse scleroderma with severe pulmonary or vascular disease. CONCLUSIONS: A subset of patients with scleroderma with antibodies to centromere and histone has been identified retrospectively, who have severe pulmonary or vascular disease. It will be of interest to follow up the clinical course of other patients with scleroderma who have both antibodies for the development of pulmonary or vascular disease.

Epitope mapping of histone 5 (H5) with systemic lupus erythematosus, procainamide-induced lupus and hydralazine-induced lupus sera.

To define the linear epitopes on H5 that react with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL) sera, concurrent overlapping hexameric peptides corresponding to the sequence of H5 were synthesized by stepwise elongation of the polypeptide chains on polyethylene supports. The hexapeptides were tested for reactivity with 8 SLE and 8 DIL sera using an enzyme linked immunosorbent assay (ELISA). SLE and hydralazine-induced lupus (HIL) antibodies were most reactive with peptide 45 (SSRQSI) and patients with procainamide-induced lupus (PIL) were most reactive with peptide 24 (SHPTYS). The epitopes of highest reactivity were in the globular domain of H5. Low reactivity was observed with carboxyl terminal peptides. These findings differ from immunoblotting studies of protease cleaved peptides which have previously shown that the H5 determinants are in the carboxyl terminus.

Human monoclonal antibodies demonstrate polyreactivity for histones and the cytoskeleton.

Systemic lupus erythematosus (SLE) and other autoimmune diseases are characterized by immune responses to intracellular, highly conserved antigens such as DNA and histone. In this study, peripheral blood lymphocytes (PBL) from a patient with histone autoantibodies were used to prepare IgM human-human hybridoma cell lines. Indirect immunofluorescence (IIF) was used to identify monoclonal antibodies that bound to cytoskeletal and other cytoplasmic constituents. These supernatants did not bind double-stranded or single-stranded DNA. However, immunoblotting revealed that 7/20 hybridomas selected for their binding to cytoskeletal components produced antibodies that also bound mammalian and avian histones. When peptide fragments of histone were used in immunoblotting experiments, it was found that the monoclonal antibodies bound to the carboxyl terminus of H1, a region previously shown to bind autoantibodies from sera of patients with SLE and drug-induced lupus (DIL). When the amino acid sequences of histones and cytoskeletal components were compared using the Swiss-Prot protein data bank, it was confirmed that there are eight regions of similarity. While the significance of polyreactive human monoclonal antibodies to cytoskeletal components and histones is not understood at present, it is possible that the human histone antibodies represent polyreactive antibodies that arise through the mechanism of molecular mimicry.

Antibodies from patients with systemic lupus erythematosus and drug-induced lupus bind determinants on histone 5 (H5).

The antigenic domains of histone 5 (H5), a highly conserved variant of histone 1 (H1), were studied in relation to their reactivity with autoantibodies found in the sera of patients with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL). While some H5 antibodies cross-react with H1, adsorption and immunoblotting studies have identified H5-specific antibodies as well. After proteolytic cleavage of H5 peptides, the reactivity of sera from these patients was tested by Western immunoblotting. All SLE (9/9) and DIL (7/7) sera bound an antigenic determinant in the carboxyl (C) terminus of H5 while none of the sera bound to the amino (N) terminus or the central hydrophobic domain. Although the reactivity of DIL sera with the purified H5 peptides was weaker than that of SLE sera, the antigenic domains bound by both groups of sera were the same. These observations demonstrate that the H5 domains reacting with DIL sera are restricted to the carboxyl terminus and are therefore no less restricted than those reacting with SLE sera. Further, the potential epitopes in the carboxyl terminus of H5 do not have a high degree of sequence identity with known mammalian peptides.

Purification of histone H1 polypeptides by high-performance cation-exchange chromatography.

Calf thymus histone 1 (H1) was cleaved by chemical and enzymatic methods and the resulting polypeptides were fractionated by high-performance cation-exchange. Up to 1 mg of H1 polypeptides were loaded onto a 50 x 5 mm I.D. cation-exchange column and fractionated to greater than 95% purity in less than 30 min. This is the first report on the separation of H1 polypeptides by a strong cation-exchange matrix. In addition, the high-performance cation-exchange chromatography protocol represents a significant decrease in fractionation time when compared to conventional ion-exchange and gel filtration chromatography. The utility of this procedure is shown when the H1 peptides purified by the protocol were used to define antigenic domains of H1 band by procainamide-induced lupus and idiopathic systemic lupus erythematosus. The majority of the sera tested by enzyme-linked immunoassay (ELISA) reacted to the C-terminal peptides of H1 indicating this to be the major antigenic domain of H1.

Antibodies to histones H1 and H5 in sera of patients with juvenile rheumatoid arthritis.

The specificity of juvenile rheumatoid arthritis (JRA) sera for histone subclasses was examined by immunoblotting. Antibodies to H1 alone were found in 4 of 21 pauciarticular-onset JRA sera, 4 of 19 polyarticular-onset JRA sera, and 2 of 11 systemic-onset JRA sera. Antibodies to H5 alone were found in 1 of 21 pauciarticular JRA sera, 1 of 19 polyarticular JRA sera, and 3 of 11 systemic JRA sera. Antibodies to both H1 and H5 were found in 4 of 21 pauciarticular JRA sera, 4 of 19 polyarticular JRA sera, and 1 of 11 systemic JRA sera. Antibodies to the core histones (H2A and H2B) were found in 1 of 21 pauciarticular JRA sera, 1 of 19 polyarticular JRA sera, and no systemic JRA sera. No reactivity to histones was observed in 30 sera from age-matched children with nonrheumatic diseases. The presence of H1 and H5 antibodies did not correlate with antinuclear antibody titers or with a homogeneous pattern of immunofluorescence. The predominance of H1 and H5 antibodies and relative absence of antibodies binding to core histones in JRA contrast with findings in adult systemic lupus erythematosus. Further, the presence of antibodies to H5 alone in some of the JRA patients indicates that the immune response in these patients is directed to determinants that are not shared by sequences of mammalian proteins.

Antinuclear, anticytoplasmic, and anti-Sjogren's syndrome antigen A (SS-A/Ro) antibodies in female blood donors.

A study of 2500 sera from female blood donors between the ages of 20 and 50 years was undertaken to determine the frequency of antinuclear (ANA), anticytoplasmic (ACA), and antimitochondrial (AMA) antibodies. When sera were tested by immunofluorescence (IF) on HEp-2 cells, 15.9 and 1.1% had ANA titers greater than 1/20 and 1/80, respectively. Analysis of these sera for autoantibody specificity showed: 1.5% antinucleolar, 1.0% anti-nuclear matrix, 0.2% anti-mitotic spindle apparatus, and 0.2% anti-primary biliary cirrhosis nuclear antigen. AMA titers of greater than 1/80 were seen in 2.5% and AMA titers greater than 1/160 were seen in 1.0%. None of the sera had anti-double stranded DNA. Testing of an additional 2500 sera for anti-Sjogren's Syndrome antigen A (anti-SS-A/Ro) revealed a frequency of 22/5000 (0.44%) with the highest frequency (0.72%) being in the 45-50 age group and a relatively high frequency (0.58%) in the 20-24 age group.