Sputum Antineutrophil Cytoplasmic Antibodies in Serum Antineutrophil Cytoplasmic Antibody-Negative Eosinophilic Granulomatosis with Polyangiitis.

RATIONALE: Eosinophilic granulomatosis with polyangiitis (eGPA) is a small-vessel vasculitis where 40% of patients present with serum antineutrophil cytoplasmic antibodies (ANCAs). We examined the presence and clinical relevance of sputum ANCAs in the serum ANCA- patients with eGPA. METHODS: ANCA was investigated in matched sputum and blood samples collected from 23 patients with eGPA (n = 10, serum ANCA+), 19 patients with eosinophilic asthma (prednisone dependent), and 13 healthy volunteers. IgG reactivity to common target antigens and cytokine profiles in sputum samples were examined. Pathogenicity of detected sputum ANCA was assessed using in vitro degranulation assays. MEASUREMENTS AND MAIN RESULTS: Most patients with eGPA (17 of 23, 74%) showed significantly increased sputum ANCAs compared with patients with eosinophilic asthma (P = 0.002) and healthy controls (P < 0.0001), irrespective of their serum ANCA status. In addition, 16 of 17 (94%) of sputum ANCA+ patients had clinical manifestations of severe asthma compared with 3 of 6 (50%) in the sputum ANCA- subset (P = 0.04). Microarray analysis of 123 common antigens failed to reveal a specific target for the ANCA IgG. However, immunoprecipitated immunoglobulins from ANCA+ sputum allowed extensive extracellular trap formations from both neutrophils and eosinophils in vitro, indicating pathogenicity of detected IgG autoantibodies. Cytokine analysis showed lung-localized increases in CXCL8 (neutrophil/eosinophil chemotaxis), CCL24 (eosinophil recruitment), and CXCL12 (lymphocyte recruitment) in the sputa from ANCA+ patients (P < 0.01). CONCLUSIONS: We report a novel finding of ANCA reactivity in the sputa of patients with eGPA in whom disease severity is driven by respiratory complications. Investigating localized autoimmunity may lead to the discovery of novel pathomechanisms, therapeutic targets, and optimal biomarkers for diagnosing and managing eGPA.

Sputum autoantibodies in patients with severe eosinophilic asthma.

BACKGROUND: The persistence of eosinophils in sputum despite high doses of corticosteroids indicates disease severity in asthmatic patients. Chronic inflamed airways can lose tolerance over time to immunogenic entities released on frequent eosinophil degranulation, which further contributes to disease severity and necessitates an increase in maintenance corticosteroids. OBJECTIVES: We sought to investigate the possibility of a polyclonal autoimmune event in the airways of asthmatic patients and to identify associated clinical and molecular characteristics. METHODS: The presence of autoantibodies against eosinophil peroxidase (EPX) and anti-nuclear antibodies was investigated in patients with eosinophilic asthma maintained on high-dose corticosteroids, prednisone, or both. The ability of sputum immunoglobulins to induce eosinophil degranulation in vitro was assessed. In addition, the associated inflammatory microenvironment in patients with detectable autoantibodies was examined. RESULTS: We report a "polyclonal" autoimmune event occurring in the airways of prednisone-dependent asthmatic patients with increased eosinophil activity, recurrent pulmonary infections, or both, as evident by the concomitant presence of sputum anti-EPX and anti-nuclear antibodies of the IgG subtype. Extensive cytokine profiling of sputum revealed a TH2-dominated microenvironment (eotaxin-2, IL-5, IL-18, and IL-13) and increased signalling molecules that support the formation of ectopic lymphoid structures (B-cell activating factor and B cell-attracting chemokine 1). Immunoprecipitated sputum immunoglobulins from patients with increased autoantibody levels triggered eosinophil degranulation in vitro, with release of extensive histone-rich extracellular traps, an event unsuppressed by dexamethasone and possibly contributing to the steroid-unresponsive nature of these eosinophilic patients. CONCLUSION: This study identifies an autoimmune endotype of severe asthma that can be identified by the presence of sputum autoantibodies against EPX and autologous cellular components.

A novel tool for B-cell tolerance research: characterization of mouse alloantibody development using a simple and reliable cellular ELISA technique.

In animal-based transplantation research, the measurement of anti-donor antibodies in transplant recipients is limited by lack of an appropriate technique. We have developed a novel immunoassay capable of quantifying antibody bound to cell-surface major histo- compatability complex (MHC) and non-MHC antigens, using splenocytes from wild-type and MHC-deficient mice as antigen-bearing target cells. We utilized our "cellular ELISA" (CELISA) technique to study the development of tolerance versus immunity in the B-cell compartment in response to neonatal exposure to allogeneic fetal liver cells (FLC). This neonatal tolerance protocol typically induces permanent acceptance of donor-type and third-party cardiac allografts, but rejection of both donor-type and third-party skin grafts occurs. C3H/He (C3H; H-2(k)) mice were injected as neonates with BALB/c (BALB; H-2(d)) FLC and transplanted as adults with C57BL/6 (B6; H-2(b)) cardiac grafts. Despite long-term acceptance of third-party B6 cardiac grafts, serum contained increased anti-B6 IgG and IgM levels as measured by CELISA; IgM production was elevated by 2 weeks posttransplant and remained stable, while IgG production increased rapidly between 2 and 5 weeks posttransplant. In another experimental setting, CELISA assays were able to detect that neonatal injection of C3H mice with FLC from wild-type B6 mice or from MHC class II-deficient or class I/II-deficient (B6 background) mice (CI(+)CII(+), CI(+)CII(-), CI(-)CII(-), respectively) prevented sensitization to B6 antigens by subsequent skin transplants but did not induce graft acceptance, whereas FLC from MHC class I-deficient-only (CI(-)CII(+)) did not prevent B6 sensitization. The CELISA technique is a simple and sensitive means for quantifying alloantibodies in mice and will assist in further delineating the role of the B-cell compartment in neonatally induced cardiac allograft acceptance.

The pancornulins: a group of small proline rich-related cornified envelope precursors with bifunctional capabilities in isopeptide bond formation.

In this report, the pancornulins are identified as members of the spr (small, proline-rich) multigene family by amino acid sequence and mass spectrometry analyses. One of the pancornulins (14.9 kDa) is identical to the protein predicted by spr-1 clone 128. The other pancornulins (16.9 kDa and 22 kDa) are novel members of the spr family. Immunoelectron microscopy of purified cornified envelopes with a pancornulin-specific antibody established these proteins more definitively as cornified envelope precursors. In addition, two-dimensional electrophoretic analyses of keratinocyte extracts labeled enzymatically with dansylcadaverine (to identify amine acceptors) or dansylPGGQQIV (to identify amine donors) showed that both glutamine and lysine residues within the pancornulins participate in the isopeptide linkage characteristic of cornified envelope formation. These results contrasted with those obtained using involucrin, a prominent cornified envelope protein shown capable of acting only as an amine acceptor in this system. Novel partial cDNAs obtained after reverse transcription and polymerase chain reaction amplification of total messenger RNA with pancornulin-specific primers suggest that the spr multigene family may be even larger than previously described. The bifunctional reactivity of the pancornulins in cross-linking and the large number of family members identified to date suggest that the pancornulins and other spr-1-related proteins may be more important in cornified envelope formation than previously considered, perhaps functioning as "bridge" molecules during the early phases of cornified envelope assembly.

O-carbamoylsalicylates: agents for modification of hemoglobins.

To combine the attractive features of cyanate and of O-acetylsalicylate as hemoglobin-modifying agents we have prepared carbamoylsalicylate. This compound is a close analogue of aspirin and also resembles a masked cyanate. O-Carbamoylsalicylate and some related carbamates modify hemoglobin substantially, even at 5 mM concentration.

Concurrent catamenial hemothorax and hemopneumothorax.

We describe a case of catamenial hemothorax and hemopneumothorax occurring on both sides simultaneously; the patient responded remarkably with danazol therapy. To our knowledge, this is previously unreported in the literature.

Asthma in the elderly: underperceived, underdiagnosed and undertreated; a community survey.

Bronchial asthma is now increasingly recognized in the elderly and is associated with significant morbidity and mortality. The aims of this study were two-fold: first, to assess the prevalence and, second, to evaluate diagnostic awareness, therapeutic management and patient perception of bronchial asthma among elderly patients in the community. From the age-sex register of an urban general practice in NE England, 2004 patients aged > 65 years were eligible for inclusion. Response to an initial screening questionnaire on respiratory symptomatology was 68% (n = 1362). Of these, 869 patients had respiratory symptoms: 390 voluntarily agreed to be evaluated further including assessment of airway physiology. In this group 369/390 had obstructive spirometry and, of these, 95 patients fulfilled clinical and physiological criteria of bronchial asthma. Prevalence of asthma within this age cohort was minimally and rather crudely assigned at 4.5% (95/2004). Among the 95 patients so-defined patients with asthma [age 70 +/- 8 years (mean +/- SD), FEV1 = 0.96 +/- 0.41, 33 male, 75 life-long non-smokers], subjective awareness, perception and attribution of pulmonary symptoms were poor. Further, despite tangible evidence of reversible and significant airflow limitation, only 21 were receiving inhaled glucocorticoid therapy (median daily dose 400 micrograms). Asthma in the elderly remains poorly perceived, poorly recognized and suboptimally treated. These findings are particularly apposite in the light of current epidemiological trends in asthma mortality and morbidity in elderly age cohorts.

Acute effects of nebulised epoprostenol in pulmonary hypertension due to systemic sclerosis.

Pulmonary hypertension often has a lethal outcome in systemic sclerosis and the treatment is challenging. Epoprostenol is a potent pulmonary vasodilator and its efficacy has been demonstrated when delivered by the intravenous and aerosolized routes. We report the haemodynamic and functional benefits of epoprostenol administered by inhalation to a spontaneously breathing patient with partially reversible pulmonary hypertension due to systemic sclerosis. Aerosolized epoprostenol, equivalent to the maximum tolerated intravenous dose (31.2 micrograms), produced a 58% fall in pulmonary vascular resistance, increased the cardiac output by 42% and improved functional performance by one MET (3.5 ml kg-1 min-1 of oxygen uptake) without any significant side-effects. Selective distribution of epoprostenol by the inhaled route may offer a new strategy for treatment of pulmonary hypertension.

Addition of intravenous aminophylline to beta2-agonists in adults with acute asthma.

BACKGROUND: Aminophylline has been used extensively in acute asthma, but its role is unclear especially with respect to any additional benefit when added to beta2-agonists. OBJECTIVES: To determine the magnitude of effect of the addition of intravenous aminophylline to beta2-agonists in adult patients with acute asthma treated in the emergency setting. SEARCH STRATEGY: Studies were identified from the following sources: The Cochrane Airways Group register (derived from MEDLINE, EMBASE, CINAHL standardised searches), hand searched respiratory journals and meeting abstracts. Potentially relevant articles were obtained, and their bibliographic lists were hand searched for additional articles. The search included searches of the database up to 1999. SELECTION CRITERIA: Randomised controlled trials comparing intravenous aminophylline versus placebo in adults with acute asthma and treated with beta-adrenergic agonists. Patients could be treated with or without corticosteroids or other bronchodilators. DATA COLLECTION AND ANALYSIS: A total of 210 abstracts were identified. Two independent reviewers selected a total of 27 eligible studies for possible inclusion, in which quality assessment was performed and a third reviewer was used to adjudicate disagreements. Peak expiratory flow (PEFR) and forced expiratory volume in the first second (FEV1) data were extracted and entered in Review Manager from these studies. Information not obtained from the authors was estimated from graphs. All data were entered and double checked by two reviewers. Results are reported as weighted mean differences (WMD) or odds ratio (OR), both with 95% confidential intervals (CI). MAIN RESULTS: Fifteen trials were included. Overall, the quality of the studies was only moderate; concealment of allocation was assessed as clearly adequate in only seven (45%) of the trials. The doses of aminophylline and other medications and the severity of asthma varied between studies. There was no statistically significant effect of aminophylline on airflow outcomes at any time period. The aminophylline treated group had higher values of PEFR at 12 (PEFR 8 L/min or 2.3%) and 24 hours (PEFR 22 L/min or 6.4%), but these were not significant (p>0.05). Two subgroup analyses were performed by grouping studies according to mean baseline airflow limitation (n = 11 studies) and the use of any steroids (n = 9 studies). There was no relationship between baseline airflow limitation nor the use of steroids on the effect of aminophylline. Aminophylline treated patients reported more palpitations/arrhythmias (OR: 2.9; 95% CI: 1.5 to 5.7) and vomiting (OR: 4.2; 95% CI 2.4 to 7.4), but no difference was found in tremor or hospital admissions. REVIEWER'S CONCLUSIONS: In acute asthma, the use of intravenous aminophylline did not result in any additional bronchodilation compared to standard care with beta-agonists. The frequency of adverse effects was higher with aminophylline. No subgroups in which aminophylline might be more effective could be identified. These results should be added to consensus statements and guidelines.

Nonasthmatic chronic cough: No effect of treatment with an inhaled corticosteroid in patients without sputum eosinophilia.

BACKGROUND: Inhaled corticosteroids are effective in suppressing a chronic cough without asthma associated with sputum eosinophilia. OBJECTIVE: To investigate the inflammatory characteristics in the induced sputum of patients with a chronic cough without asthma or known cause and the effects of budesonide treatment on chronic cough in those patients. PATIENTS AND METHODS: Forty-four adults (mean [minimu, maximum] age of 45 years [20,75], 28 women, 17 atopic subjects and 32 nonsmokers], with a daily bothersome cough for at least one year and who had no evidence of asthma or other known cause for the cough, were consecutively enrolled. The trial was a randomized, double-blind, controlled parallel group trial of budesonide 400 mg twice daily for two weeks versus placebo. Patients then received open administration of the same dose of budesonide for a further two weeks. Sputum was induced before and at the end of each treatment period. Cough severity was documented by a visual analogue scale. RESULTS: Thirty-nine (89%) patients produced mucoid sputum after induction on at least one study visit. At baseline, the majority (59%) had a mild elevation in the median proportion of neutrophils (65%). All had elevated fluid phase levels of fibrinogen (3200 mg/L) and albumin (880 mg/L), and high levels of interleukin-8 and substance P. Interleukin-8 correlated with neutrophils (rho=0.72, P<0.001), fibrinogen (rho=0.65, P<0.001), albumin (rho=0.67, P=0. 001) and eosinophil cationic protein (rho=0.60, P=0.001). Substance P correlated with albumin (rho=0.60, P=0.006). No subject had an increase in eosinophils. Treatment with budesonide did not affect cough or sputum measurements. CONCLUSIONS: Patients with nonasthmatic chronic cough enrolled in this study had evidence of a mild neutrophilia and/or microvascular leakage. Chronic cough did not respond to treatment with budesonide, perhaps because the cause was not associated with sputum eosinophilia.

Sputum cell counts and exhaled nitric oxide in patients with gastroesophageal reflux, and cough or asthma.

BACKGROUND: Gastroesophageal reflux (GER) is commonly associated with chronic cough and asthma, but there is little or no information on the nature of any associated airway inflammation. OBJECTIVE: To observe whether the association with GER worsens airway inflammation in patients with chronic cough or asthma. PATIENTS AND METHODS: The airway inflammatory indexes in induced sputum and exhaled air were examined in a cross-sectional study of 11 patients with cough and GER, nine patients with mildly symptomatic asthma and GER, nine patients with mildly symptomatic asthma without GER and nine normal, healthy control subjects. GER was shown objectively by 24 h ambulatory pH recording. RESULTS: The sputum total cell count, the proportion of neutrophils and macrophages, and the fibrinogen level were normal in all four groups, with no significant differences among the groups. The sputum eosinophil and metachromatic cell percentages, and eosinophil cationic protein levels were normal in patients with cough and GER. They were significantly increased in patients with asthma compared with healthy subjects (P<0.01) and patients with cough (P<0.01), but were not different between groups with and without GER. Exhaled nitric oxide levels showed similar results (P<0.01). The correlations between the number of episodes of reflux and the proportion of sputum eosinophils, neutrophils or exhaled nitric oxide were modest but not significant. CONCLUSIONS: GER, when associated with cough or mildly symptomatic asthma, does not cause or aggravate existing airway inflammation as measured by induced sputum cell counts and fibrinogen level, or by exhaled nitric oxide.