Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS: We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS: Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS: Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Development and implementation of guidelines in allergic rhinitis ­ an ARIA-GA2LEN paper.

The links between asthma and rhinitis are well characterized. The Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines stress the importance of these links and provide guidance for their prevention and treatment. Despite effective treatments being available, too few patients receive appropriate medical care for both diseases. Most patients with rhinitis and asthma consult primary care physicians and therefore these physicians are encouraged to understand and use ARIA guidelines. Patients should also be informed about these guidelines to raise their awareness of optimal care and increase control of the two related diseases. To apply these guidelines, clinicians and patients need to understand how and why the recommendations were made. The goal of the ARIA guidelines is to provide recommendations about the best management options for most patients in most situations. These recommendations should be based on the best available evidence. Making recommendations requires the assessment of the quality of available evidence, deciding on the balance between benefits and downsides, consideration of patients' values and preferences, and, if applicable, resource implications. Guidelines must be updated as new management options become available or important new evidence emerges. Transparent reporting of guidelines facilitates understanding and acceptance, but implementation strategies need to be improved.

Allergenic fungi in allergic fungal sinusitis.

BACKGROUND: Patients with allergic fungal sinusitis demonstrate skin test reactivity to many fungal extracts. Various fungi have been isolated from the characteristic allergic mucin. OBJECTIVE: This study was designed to identify allergens in allergic mucin and to compare them to those found in commercial fungal extracts. METHODS: Allergic mucin was collected during functional endoscopic sinus surgery from 11 patients meeting strict diagnostic criteria for allergic fungal sinusitis and from three allergic rhinitis patients with chronic sinusitis. A portion was solubilized in saline and centrifuged. To identify allergens, proteins in allergic mucin and fungal extracts were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose and immunostained using patient sera and enzyme-labeled anti-human IgE. RESULTS: All patient sera recognized numerous bands ranging from 18 to 90 kD. In mucin, bands were consistently found in the 35 to 50 kD range. Corresponding bands in fungal extracts were found in only 1/11 patients with allergic fungal sinusitis. Sera from 4/11 patients detected an 18-kD protein in allergic mucin, but sera from all patients with allergic fungal sinusitis recognized an 18-kD protein in commercial fungal extracts. Sera from selected patients with allergic fungal sinusitis detected human epithelial proteins in the 35 to 50 kD range. CONCLUSIONS: Fungal allergens were not detected in allergic mucin of all patients with allergic fungal sinusitis. The 18-kD allergen appears to be shared by many fungi, and may be a fungal panallergen. The source of the apparent allergens in the 3550-kD range warrants further study.

Local and systemic eosinophil activation in allergic fungal sinusitis.

BACKGROUND: In allergic fungal sinusitis diagnostic and monitoring criteria are not firmly established, and the role of eosinophils in pathogenesis is not clear. OBJECTIVE: To determine whether assessment of eosinophil activation by measurement of eosinophil cationic protein in serum or allergic mucin would be useful in distinguishing patients with allergic fungal sinusitis from patients with chronic sinusitis of other etiologies. METHODS: Thirteen patients referred for possible allergic fungal sinusitis were evaluated and given a definite allergic fungal sinusitis diagnosis if they met five of the following six criteria: (1) history and physical not suggesting another etiology, (2) sinus computed tomography consistent with allergic fungal sinusitis, (3) typical allergic mucin, (4) fungus isolated from allergic mucin, (5) presence of fungal-specific IgE, and (6) elevated total IgE. Eosinophil cationic protein, a marker of eosinophil activation, was measured in serum and allergic mucin. RESULTS: Nine patients met criteria for allergic fungal sinusitis. All patients had nasal polyps and were atopic. Eight of the patients had allergic rhinitis and three had asthma. Mean total IgE at surgery was 1,385 IU/mL. A fungus was isolated from allergic mucin of eight patients. All patients demonstrated fungal-specific IgE. Mean allergic mucin eosinophil cationic protein levels obtained at surgery were significantly higher in patients with allergic fungal sinusitis than in four patients not meeting strict diagnostic criteria, and in 16 control patients having sinus surgery for other indications. There was no significant difference in serum eosinophil cationic protein levels between the three groups. Serial allergic mucin eosinophil cationic protein levels appeared to correspond with disease activity in some allergic fungal sinusitis patients. CONCLUSIONS: Eosinophils in allergic mucin are activated. Measuring eosinophil cationic protein may be useful in diagnosis of allergic fungal sinusitis and in monitoring response to therapy.

Use of thiocyanate elution to estimate relative avidity of allergen specific IgE antibodies.

Although several methods for estimating avidity of antigen-antibody reactions are available, most are impractical for the study of human IgE antibodies because of a requirement for pure allergen and antibody in relatively large amounts. To determine the relative avidity of specific IgE antibodies for Dermatophagoides pteronyssinus allergens, seven concentrations of the chaotropic thiocyanate ion were used to disrupt epitope-antibody binding in a specific IgE immunoassay system, using sera from 16 allergic patients with marked skin test reactivity to a standardized D. pteronyssinus extract. Relative avidity, the molarity of thiocyanate required to produce a 50% decrement in binding, ranged from 0.29-3.1. Within assay coefficient of variation (CV) was 9.9% and between assay CV was 13%. D. pteronyssinus specific IgE levels ranged from 0.66-141 kUa/L, not correlating with relative avidity (rho = -0.12). Thiocyanate elution appears to be a useful method for estimating relative avidity of specific IgE antibodies for the myriad epitopes of the allergenic proteins in an allergen extract. It could be used to study the immunochemistry of specific IgE assays; avidity maturation in allergen immunotherapy and in asymptomatic but sensitized patients; and preseasonal versus postseasonal changes in avidity within individuals. With a suitable solid phase, it could be modified to examine avidity at the epitope level.

Asthma as an inflammatory disease: implications for management.

BACKGROUND: Eosinophilic inflammation plays a central role in the pathogenesis of asthma. Striking inflammatory changes are present in the airways of patients with all levels of disease severity. The degree of airway inflammation correlates with airway hyperresponsiveness, the primary physiologic abnormality of asthma. Inflammation is typically initiated by immunologic events (including allergy) and is driven by mediators released by various cells of the immune system, particularly eosinophils, monocytes and macrophages, lymphocytes, and mast cells. METHODS: Literature on asthma and the inflammatory response was drawn from recent articles presented and reviewed in journal clubs and from selected articles from the National Library of Medicine. RESULTS AND CONCLUSIONS: The inflammatory process can be divided into six steps: triggering, signaling, migration, inflammatory cell activation, tissue damage, and resolution. Recognition of the importance of inflammation in the pathogenesis of asthma and the progression of the disease has shifted research efforts and the development of new therapeutic agents toward reduction of airway inflammation. Anti-inflammatory therapy, which can be directed against specific steps in the inflammatory process, actually reduces bronchial hyperresponsiveness. Although anti-inflammatory management has assumed a primary role in asthma therapy, short acting beta 2-adrenergic receptor agonists are needed for treatment of acute symptoms, and some patients require regular beta 2-agonist therapy despite apparently adequate anti-inflammatory therapy.

Fungal sinusitis: an update.

OBJECTIVE: To review the classification of fungal sinusitis as well as discuss current approaches to diagnosis and management. DATA SOURCES: A MEDLINE literature search was performed using the index terms sinus infection, fungal, diagnosis, radiology, microbiology, and treatment. The search was restricted to the English language and human subjects. With one exception the references were restricted to the last 10 years. Clinical data from studies performed at our institution were also included. RESULTS: Fungal sinusitis can be divided into four primary categories: (1) acute/fulminant (invasive), (2) chronic/indolent (invasive), (3) fungus ball, and (4) allergic fungal sinusitis. Each subtype has unique immunologic, pathologic, and clinical features. Allergic fungal sinusitis is the most recently described and most common form. The treatment and prognosis of fungal sinusitis varies significantly among the four different categories. CONCLUSION: Recent advances in endoscopy and computed tomography have enhanced the understanding of fungal sinusitis; however, they remain diseases surrounded by controversy. New insights into the etiology and pathogenesis of these diseases along with advances in diagnosis and treatment will lead to improved medical therapy.

Laboratory evaluation of a commercial immunoassay for fire ant allergen-specific IgE antibodies.

BACKGROUND: In vitro testing for fire ant sensitization would be useful for research purposes and in special clinical situations. METHODS: Laboratory performance of a commercial assay (Pharmacia CAP System, [PCS]), for specific IgE to Solenopsis invicta whole body extract was studied in 46 persons. Assay results were compared with those of venom skin testing, RAST, and ELISA. The manufacturer's global cutoffs were compared with cutoffs set by using methods derived from analytical detection limit theory. RESULTS: Thirty-two study subjects had positive skin test results, and 14 had negative results. Raw PCS data demonstrated a high level of correlation with RAST (rho = 0.941) and ELISA (rho = 0.931), and showed good correlation with skin testing (rho = -0.769). Analysis of binormal receiver operating characteristic curves, using skin test results as the reference standard, demonstrated no difference in performance among the three assays. The fixed global quantitative cutoff of 0.35 kUa/L was relatively insensitive. Use of the manufacturer's qualitative alternate scoring method cutoff substantially increased sensitivity without loss of specificity, as did lower limit of detection set by use of diluent. CONCLUSIONS: In situations in which skin testing for fire ant sensitization is not feasible, PCS appears to be an acceptable in vitro alternative method for determination of fire ant allergen-specific IgE.

The beta-2 adrenergic receptor and its agonists.

The beta 2 adrenoceptor has been cloned and sequenced, and details of how agonist stimulation ultimately results in clinically evident effects are now known. Receptor desensitization by agonist is an almost universal process that can be prevented and reversed by corticosteroids. Although immunologic mechanisms appear to prevail in asthma, beta 2 receptor dysfunction may be important in some circumstances. Many receptor agonists are available for asthma therapy, including a new generation of long acting agents (such as salmeterol) with duration of action of 12 hours or more. Statistical links between beta agonist use and asthma mortality or morbidity warrant careful examination for clinical relevance, but may not be dismissed. Beta agonists are relatively safe and clearly effective in asthma, but anti-inflammatory management (including allergen avoidance and immunotherapy when appropriate) is indicated in patients who require chronic therapy.

Detection limits and receiver operating characteristic curve analysis in the evaluation of specific IgE assays.

One of the major sources of uncertainty and controversy in allergy testing is the definition and clinical significance of a positive test result in the low assay range. Use of analytical detection limit theory can guide the diagnostic allergy laboratory director in setting a lower assay cutoff and in the evaluation of the recommended cutoff by the manufacturer of the assay. Several methods for calculating lower limit of detection are applicable to specific IgE assay methods. Receiver operating characteristic (ROC) curves illustrate the relationship between statistical sensitivity and specificity as cutoffs are adjusted, and statistical analysis of ROC curves is an invaluable aspect of the comparative performance evaluation of two or more assays relative to an independent standard such as skin testing.

Aerobiology of the Colorado Rockies: pollen count comparisons between Vail and Denver, Colorado.

Pollen patterns were compared between Vail, CO (8,200 feet elevation), Aspen, CO (7,900 feet) and Denver, CO (5,280 feet) from 1984 through 1988. Counts were obtained at all sites with a volumetric intermittent cycling rotating impaction sampler. Aspen and Denver were compared in 1984, and Vail and Denver from 1985 through 1988. While counts were generally lower in the mountain sites than Denver, certain pollens, especially trees, were quite high. Ragweed was essentially absent from Aspen and Vail, and chenopod-amaranth counts were very low. Cedar, pine, and aspen frequently pollinated despite active snowfall.

Immunoassay of specific IgE: low level assays require measurement of allergen specific assay background.

The allergen-specific backgrounds of a paper disk radioimmunoassay (RIA) system and a cellulose sponge fluorescent enzyme immunoassay (FEIA) system were evaluated using three inhalants, timothy, short ragweed, and cat, with reagents obtained from the same manufacturer. Radioimmunoassay was performed with Phadebas RAST reagents by the modified RAST method, and FEIA by the Pharmacia CAP System. Horse serum, 5% HSA, assay diluent, and serum pools from nonallergic and allergic patients, were assayed. The lower limit of detection (LLD) was defined using both the Z distribution (as is conventional) and the t distribution. The solid phases, analytes, and assays differed (p less than .001) in background results. For RIA, background was lowest for timothy and highest for cat; for FEIA, background was lowest for cat and highest for short ragweed. For RIA, background assessed with the allergic serum pool was higher than the other analytes; for FEIA, responses of the five analytes did not differ. For timothy and short ragweed, background of RIA was lower than FEIA. For FEIA, the highest LLD calculated using the Z distribution was 11 SD lower than the manufacturer's recommended quantitative cutoff; for RIA, the highest LLD calculated was 2.5 SD higher than the recommended analytic cutoff. The analytic false positive rate for RIA may differ between allergic and nonallergic patient populations. Laboratories reporting results near either assay's background should set LLD based on assay of allergen-specific negative controls in each assay run.

Immunoassay of specific IgE: use of a single point calibration curve in the modified radioallergosorbent test.

Interest in immunoassay standardization has prompted development of specific IgE assays reporting results related to the international IgE reference. To examine the single point calibration curve employed in the modified RAST assay (MRT) to convert MRT counts to IgE units, independent dilutions of a 25 kU/L total IgE reference and nine allergic sera (three each for short ragweed, cat, and timothy) were made in horse serum and assayed. In a log-log plot, the single point curve was, by definition, linear over its entire range; the dilution curve was curvilinear because of reagent system saturation, which was at 7 kU/L. Curves were not parallel (P less than .001). Allergen-specific dilution curves showed saturation points at values similar to or less than the total IgE system. The linear portions of these curves paralleled the total IgE dilution curve but not the single point curve. This lack of parallelism would have resulted in varying magnitudes of error in estimation of IgE antibody levels in the upper and lower assay ranges, and would imply a lower detection limit for IgE than that which the assay actually has. Modified RAST assay is not appropriate in research or a clinical situation in which accurate quantitative results are needed. Modified RAST assay would furthermore be an inappropriate means of assigning units to proposed reference preparations for standardization.