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.

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.

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.

A double-blind evaluation of skin test suppression produced by two doses of terfenadine.

For some patients, terfenadine, in the currently recommended dose of 60 mg twice daily (bid), may be only modestly effective in the treatment of allergic rhinitis. In a double-blind placebo-controlled crossover study of 12 patients, a larger dose (300 mg bid) was evaluated for its suppression of titrated skin tests to histamine and compound 48/80 to determine whether this regimen might result in greater suppression while it maintained the freedom from side effects of the presently recommended dose. In seven patients, skin test suppression by these two doses of terfenadine each administered for 3 days, was compared to that produced in an earlier study by 3 days of treatment with chlorpheniramine (8 mg three times a day). The 300 mg bid terfenadine regimen produced significantly greater skin test suppression (p less than 0.05) than the currently recommended 60 mg bid dose. There was no significant difference in side effects between the two doses, and neither active treatment regimen produced more side effects than placebo treatment. Both doses of terfenadine suppressed cutaneous reactivity significantly more than had chlorpheniramine. It is concluded that the presently recommended dose of terfenadine produces submaximal skin test suppression and that further studies are needed to investigate the clinical efficacy and safety of larger doses of terfenadine in the treatment of allergic rhinitis.

The hidden tonsils of Waldeyer's ring.

The visible and the hidden tonsils of Waldeyer's ring merit attention from specialists in allergy and clinical immunology not only because they are sites of antigen recognition and synthesis of antibody, including IgE, but also because they may produce a wide range of symptoms in the upper airway. Ordinarily, such symptoms may be differentiated from those of classic allergic rhinitis, but allergists commonly see patients with a variety of upper airway complaints that on investigation prove not to have an allergic basis. Use of flexible fiberoptic upper airway endoscopy, a simple office procedure that permits direct inspection of the recessed areas of the upper airway, will result in timely evaluation and treatment of pathology of these upper airway lymphatic structures, and identification of patients requiring referral to another specialist.

Comparison of skin testing and three in vitro assays for specific IgE in the clinical evaluation of immediate hypersensitivity.

A new assay, Pharmacia CAP System (PCS), for allergen-specific IgE (sIgE) was evaluated in 198 new patients presenting with respiratory symptoms to an urban allergy practice. An experienced allergist examined each patient and clinically assessed sensitivity to timothy, short ragweed, Alternaria tenuis, cat, or D. farinae. Puncture and selected intracutaneous skin tests (ST) with these inhalant extracts were then performed. The physician again rated the likelihood of clinical sensitivity to each inhalant, and serum was obtained for sIgE measurements by Phadebas RAST, modified RAST, and PCS. Results of the three in vitro tests (IVT) correlated well with each other and generally agreed with physician assessments and ST results. Individual differences for extracts and assay methods were identified. A few patients with negative ST had positive IVT, but more patients with positive ST were negative by IVT. Modified RAST had greater sensitivity but less specificity than the other two IVT. Analysis of receiver operating characteristic curves showed that sensitivity of the three assays when compared at the 95% level of specificity, did not differ. This result suggests that the cutoff criterion for a positive modified RAST result is too low and should be reevaluated. Skin tests remain the most sensitive and specific test available. The Pharmacia CAP System is a clinically useful assay for sIgE and appears to be a clear advancement for IVT technology.

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.

Chronic chlorpheniramine therapy: subsensitivity, drug metabolism, and compliance.

To investigate whether patients develop true subsensitivity to antihistamines during chronic therapy, we studied 14 adult subjects who received chlorpheniramine for 3-day and 3-week trials of therapy. Titrated skin tests to histamine and compound 48/80, chlorpheniramine blood levels (by HPLC), compliance, and side effects were monitored and compared during the two courses of therapy and their respective 72-hour washout periods. We found a significant correlation between chlorpheniramine blood levels and skin test suppression during both the 3-day and 3-week therapies. The 3-day chlorpheniramine therapy was more clinically effective (measured by skin test suppression corrected for serum chlorpheniramine concentration) than the 3-week therapy (P less than .01). Chlorpheniramine serum half-lives and 2-hour chlorpheniramine blood levels were not significantly different after the 3-day and 3-week trials. Compliance was significantly worse (P less than .01) during 3-week therapy. Medication side effects (particularly drowsiness) were frequently reported during both courses of therapy. We conclude that subsensitivity to chlorpheniramine does develop in adult patients receiving 3 weeks of therapy. This subsensitivity is not explained by changes in drug metabolism. In addition to subsensitivity, poor compliance may contribute to sub-therapeutic results during chronic antihistamine therapy. Side effects from antihistamines may also require individualization of therapy for certain patients.

Use of standardized and conventional allergen extracts in prick skin testing.

This study examined whether commercially available conventional and standardized allergen extracts differ enough in potency to affect routine prick skin test results. Extracts of white oak, timothy, Bermuda, Russian thistle, short ragweed, sagebrush, Alternaria, and cat dander were examined in allergic patients and in nonatopic subjects with no personal or family history of asthma, rhinitis, or eczema. Conventional nonstandardized extracts (1:10 or 1:20 wt/vol) from two sources were compared with three concentrations (100,000, 10,000, and 1000 AU/ml) of a single standardized extract. Preparations were compared in the allergic patients with computerized planimetry, and in all patients and subjects with a conventional skin test grading system. Skin test area for the conventional extracts generally fell between the 10,000 and 100,000 AU/ml concentrations of the standardized extract. Skin test reactivity to at least one allergen extract occurred in 31% of the nonatopic subjects; there was no difference between the number of 3+ and 4+ reactions for conventional and standardized extracts. Results indicate that standardized and conventional extracts are frequently similar, but are not directly interchangeable.

The histamine content of allergen extracts.

The histamine content of 108 inhalant, food, and venom extracts from four commercial sources was measured by chemical (glass fiber-based) and immunologic (competitive RIA) methods. Histamine was present in 64 of 76 inhalant extracts (range, 0.005 to 7.4 micrograms/ml), 20 of 26 food extracts (range, 0.16 to 23 micrograms/ml), and six of six venoms, 100 micrograms/ml (range, 1.0 to 38 micrograms/ml). Histamine was removed by treatment with diamine oxidase or dialysis of an extract. Repeat assay of selected extracts after addition of known amounts of histamine in the glass fiber-based method produced additive results, and glycerin- or phenol-extract preservatives did not affect assay performance. Timed extractions of dried-pollen grains demonstrated maximal histamine content at 30 seconds, suggesting that histamine is an inherent component of at least some pollens. Histamine found in some allergen extracts could, under extreme circumstances, produce false-positive results in skin testing and in basophil histamine release assays, and could affect the result of research that uses intact pollen or allergen extracts.

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.

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.

Evaluation of nasal patency by fiberoptic rhinoscopy.

A novel method of assessing anterior nasal patency by flexible fiberoptic rhinoscopy was evaluated in two centers by comparison with active anterior rhinomanometry. Rhinoscopy and rhinomanometry were performed 20 times on each of 14 subjects during 14 to 26 minutes. The procedure was videotaped, and nasal airway area was measured from a video monitor. The mean coefficients of variation for rhinoscopy and rhinomanometry were 14% and 19%, respectively. Another investigator examined five subjects by rhinoscopy 30 times in 15 minutes; the mean coefficient of variation was 9%. These means were not significantly different. Rhinoscopy and rhinomanometry were performed at 30-minute intervals for a 6-hour period in 13 subjects. Three investigators independently evaluated results for changes in nasal function characteristic of the nasal cycle. Rhinoscopy detected the nasal cycle in an average of 72% of subjects and rhinomanometry in 49%. Both methods detected the response to topically applied oxymetazoline and methacholine. Results indicate that, whereas the two procedures evaluate different aspects of nasal function, their precision is similar. Rhinoscopy additionally permits evaluation of nasal mucosal changes occurring from disease or treatment.

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.

Anaphylaxis to piñon nuts.

A 21-year-old white male developed life threatening systemic anaphylaxis within seconds of ingesting a small amount of a cookie containing piñon nuts. Skin testing, ELISA, and basophil histamine release studies demonstrated piñon nut-specific IgE. Electrophoresis of the piñon nut extract demonstrated 30 bands, three of which (in the 66 to 68,000 dalton range) bound IgE in the patient's serum in an immunoblot. Ingestion challenge was not performed due to the severity of the patient's reaction. Although used for centuries in certain cultures, piñon nuts are now being eaten more frequently in the American diet. Physicians should be aware of the potential for anaphylactic reactions following ingestion of this food.

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.

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.