Similar biological activity in skin prick test for Oralair® (8200 BAU) and Grazax® (6200 BAU) reinforces effective SLIT dosing level.

In Europe, allergen extracts are standardized based on skin prick wheal size in 20-30 allergic subjects. To understand the biological activity of clinically effective Sublingual immunotherapy, we used this method to determine the biological activity of solution and tablet Timothy grass pollen (TIM) extracts, compared to an FDA-approved extract (Reference) of 10 000 BAU/ml. Blinded, quadruplicate skin prick tests with concentrate and three serial half-log dilutions allowed the construction of a semilogarithmic regression line per extract. Bioequivalent allergy units (BAU) values were obtained from the comparison with reference. Extracts and dilutions showed a neat linear dose response (all: R2 > 0.98) in 33 rhinitis patients. Relative potencies: Staloral® 12 000 BAU/ml, Soluprick® 10 300 BAU/ml, Oralair® 8200 BAU, and Grazax® 6200 BAU. Even though all extract concentrates differed in wheal size (P = 0.01-0.001), Grazax® producing a 25% smaller wheal size than Oralair® , and the biological activity of these clinically effective TIM tablets led in the same range (6200-8200 BAU; 0.92-1.23 cm2 ). SLIT dose-finding studies for other pollens might start with allergen extracts producing 1.1 cm2 wheal surface.

Endotoxin contamination of engineered nanomaterials.

Endotoxin has established health impacts and may be a potential confounding factor in toxicity studies of engineered nanomaterials (ENM). We aimed to characterize endotoxin contamination for a representative set of carbon-based ENM. The established method for quantifying endotoxin relies on its activity in a complex biochemical assay system. Because of their physical and chemical properties, measurement of endotoxin associated with many ENM presents non-trivial technical challenges. We have made progress in identifying and implementing methods for ENM analysis with respect to endotoxin content, revealing varying levels of endotoxin contamination in the ENM examined here. The physical association of ENM and endotoxin and their shared physiological effects suggest the possibility that contaminating endotoxin may contribute to the toxicity that is ascribed to ENM. We found in this small number of samples that endotoxin levels were not related to type of ENM or surface area but may be introduced randomly during manufacture.

A comparison of in vitro potency between European and Mexican allergen extracts and US (CBER/FDA) reference extracts.

BACKGROUND: The most important allergen manufacturers are based in Europe and in the US. In some countries local products are also sold. No comparison between European, US and local products has been made until now. AIM OF THE STUDY: To determine total protein content and total specific IgE binding capacity or major allergen content of diagnostic extracts from European, US and Mexican origins relative to the CBER/FDA reference extracts for Dermatophagoides pteronyssinus (Dpt), Bermuda grass and cat (10,000(B) AU/mL). METHODS: Diagnostic extracts were purchased from various manufacturers, blinded and shipped to the analysing laboratory, where the following assays were conducted: total protein concentration (Bradford), specific IgE competition ELISA (Dpt and Bermuda grass) and determination of Fel d 1 U/mL. When available, CBER/FDA recommended tests and reagents were used. RESULTS: Total protein content of US reference extracts was higher than all other extracts. Relative potency of European and US-bought Dpt extracts 3,300-4,400 AU/mL, Bermuda grass 800-2,500 BAU/mL and cat 2.1-4.4 Fel d IU/mL (Ref. 19 U/mL), with one exception. Locally produced Mexican products were almost all below 1,000 (B)AU/mL. CONCLUSIONS: Three diagnostic extracts from European manufacturers and from Mexican providers which obtain extracts in US have a <50% relative potency compared to 10,000 (B)AU/mL US extracts. Locally produced Mexican extracts have much lower total protein content and specific IgE binding capacity. These in vitro results must be complemented with other in vitro and in vivo skin prick tests to obtain a more complete picture of comparison of potency. Nevertheless results are quite consistent for the allergens tested here.

Pheromone-induced degradation of Ste12 contributes to signal attenuation and the specificity of developmental fate.

The Ste12 transcription factor of Saccharomyces cerevisiae regulates transcription programs controlling two different developmental fates. One is differentiation into a mating-competent form that occurs in response to mating pheromone. The other is the transition to a filamentous-growth form that occurs in response to nutrient deprivation. These two distinct roles for Ste12 make it a focus for studies into regulatory mechanisms that impart biological specificity. The transient signal characteristic of mating differentiation led us to test the hypothesis that regulation of Ste12 turnover might contribute to attenuation of the mating-specific transcription program and restrict activation of the filamentation program. We show that prolonged pheromone induction leads to ubiquitin-mediated destabilization and decreased amounts of Ste12. This depletion in pheromone-stimulated cultures is dependent on the mating-pathway-dedicated mitogen-activated protein kinase Fus3 and its target Cdc28 inhibitor, Far1. Attenuation of pheromone-induced mating-specific gene transcription (FUS1) temporally correlates with Ste12 depletion. This attenuation is abrogated in the deletion backgrounds (fus3Delta or far1Delta) where Ste12 is found to persist. Additionally, pheromone induces haploid invasion and filamentous-like growth instead of mating differentiation when Ste12 levels remain high. These observations indicate that loss of Ste12 reinforces the adaptive response to pheromone and contributes to the curtailing of a filamentation response.

A family of destabilized cyan fluorescent proteins as transcriptional reporters in S. cerevisiae.

The 'programmable' features of the N-end rule degradation pathway and a ubiquitin fusion strategy were exploited to create a family of destabilized cyan fluorescent proteins (CFP) to be used as transcriptional reporters. The N-degron CFP reporters characterized in this report have half-lives of approximately 75, 50 and 5 min, but further modification of the N-degron signal sequences could readily generate additional variants within this range. These destabilized CFP reporters have been engineered into convenient plasmid constructs with features to enable their expression from upstream activating sequences of choice and to facilitate their targeted integration to the URA3-TIM9 intergenic region of chromosome V. The advantages and limitations of these reporters as temporal indicators of gene expression in living cells are illustrated by their application as reporters of galactose- and pheromone-induced transcription. The plasmid design we describe and the range of different stabilities that are theoretically feasible with this strategy make the N-degron CFP reporters easily adapted to a variety of applications.

Pheromone induction promotes Ste11 degradation through a MAPK feedback and ubiquitin-dependent mechanism.

Ste11 is the mitogen-activated protein kinase (MAPK) kinase kinase in the MAPK cascades that mediate mating, high osmolarity glycerol, and filamentous growth responses in Saccharomyces cerevisiae. We show stimulation of the mating pathway by pheromone promotes an accelerated turnover of Ste11 through a MAPK feedback and ubiquitin-dependent mechanism. This degradation is pathway specific, because Ste11 is stable during activation of the high osmolarity glycerol pathway. Because the steady-state amount of Ste11 does not change significantly during pheromone induction, we infer that maintenance of MAPK activation involves repeated cycles in which naive Ste11 is activated and then targeted for degradation. This model predicts that elimination of active Ste11 would rapidly curtail MAPK activation upon attenuation of the upstream signal. This prediction is confirmed by the finding that blocking ubiquitin-dependent Ste11 degradation during pheromone induction abolishes the characteristic attenuation profile for MAPK activation.

Allergen immunotherapy.

Allergen immunotherapy plays an important role in the treatment of allergic diseases and asthma. This article is a brief review of the current approaches, including patient and allergen selection, routes of administration, and use of standardized allergen vaccines. New approaches offering potentially useful strategies based on recent studies of T-cell epitopes, cytokines, and anti-IgE and DNA vaccines also are considered.

Isolation and molecular characterization of gibberellin-regulated H1 and H2B histone cDNAs in the leaf of the gibberellin-deficient tomato.

After differential screening we isolated cDNA clones encoding a histone H1 (leH1) and three variants of histone H2B (leH2B-1, -2 and -3) from the gibberellin (GA)-deficient mutant of tomato (gib-1). The deduced polypeptide of leH1 is 271 amino acids long and exhibits the typical tripartite structure of histones H1. The full-length cDNA clone leH2B-1 encodes for a protein of 142 amino residues and shows the tripartite organization of histones H2B. The histones leH1 and leH2B, which show no tissue specificity, are developmentally expressed in the leaf. The mRNA accumulation was higher in organs which contain meristematic tissue and/or which have a high proportion of actively cycling cells. In the leaf of the gib-1 mutant we demonstrated GA-enhanced histone leH1 and leH2B expression which was not observed in the wild type. GAs of the early-13-hydroxylated pathway (GA1 and GA3) caused most enhanced transcription compared to GAs of the early-non-hydroxylation pathway (GA4 and GA9). Application of GA to the mutant increased histone expression that could correlate with enhanced DNA replication in leaf tissue. Increased chromosome replication may indicate that there is a higher rate of cell division and/or increase of endopolyploidy which both may be dependent on cell elongation induced by GAs.

IgE-sensitization to cellular and culture filtrates of fungal extracts in patients with atopic dermatitis.

BACKGROUND: Patients with atopic dermatitis may experience exacerbations of eczema triggered by various inflammatory stimuli. One mechanism may be IgE-mediated reactions to dermatophytes since these patients are more likely to acquire skin infections with dermatophytes and may become sensitized. OBJECTIVE: This study investigates IgE-sensitization to fungi in patients with atopic dermatitis and compares the biologic activity of culture filtrates and cellular fungal extracts. The following allergen extracts were provided as culture filtrates and cellular extracts: Candida albicans, Fusarium moniliforme, and Penicillium notatum. In addition, Pityrosporum ovale and Trichophyton rubrum cultures were included in the test panel. METHODS: Fifteen patients with clinical findings suggesting dermatophytosis and 11 controls were selected. Each subject was tested by leukocyte histamine release and skin prick test to each fungal extract. The extracts were separated and reduced by sodium dodecylsulfate polyacrylamide gel electrophoresis and analyzed by IgE-immunoblotting with sera from all study subjects. RESULTS: Fourteen patients (93%) reacted to one or several fungal extracts by releasing histamine when challenged in vitro. By immunoblotting experiments, patient sera showed binding to a wide range of components in all extracts. Patient sera recognized allergenic components shared by culture filtrates and cellular extracts but with higher frequent and greater intensity in culture filtrates. Although culture filtrates generated more frequent and potent IgE-reactions than the cellular extracts, the difference was not statistically significant. Biologic potency was similar when evaluated by skin prick tests and leukocyte histamine release. CONCLUSION: Patients with atopic dermatitis may develop specific IgE-antibodies to a number of fungi as demonstrated by IgE-immunoblotting. In selected patients, fungi may trigger an IgE-mediated reaction that may contribute to the exacerbation of eczema. Approximately, one-half of the patients, however, produced IgE-antibodies to fungal (glyco)proteins without a significant histamine release or skin test response possibly because of nonspecific interaction with carbohydrate moieties on IgE and poor biologic activity of IgE antibodies directed to cross-reactive carbohydrate determinants of fungal glycoproteins. This warrants caution when interpreting clinical relevance of serologic measurements of fungal IgE-antibodies.

Mapping of an allergenically important determinant of grass group I allergens.

This report documents the mapping of the second major epitope, previously described as site D, of grass group I allergens to residues 23-35 of meadow fescue group I (STFYGKPTGAGPK). Mapping was accomplished by screening fractions from a meadow fescue group I tryptic digest for peptide(s) that inhibit the ability of monoclonal antibody 24.64 (specific for site D) to bind to immobilized group I allergen. One such peptide, representing residues 23-35 of meadow fescue group I, was identified. Additional studies with the use of synthetic analogs of this peptide demonstrate that it binds mAb 24.64 directly. Examination of extracts containing group I glycoproteins from seven other species of grass confirms antigenic cross-reactivity due to this peptide. We also report based on protein sequence analysis that the amino terminal segment (which includes the site D epitope) of GpI allergens from seven different grass species is highly conserved and contains two hydroxyproline residues and an N-linked carbohydrate moiety.

Effects of adding behavioral treatment to opioid detoxification with buprenorphine.

This trial assessed whether behavioral treatment improves outcome during a 26-week outpatient opioid detoxification. Thirty-nine opioid-dependent adults were assigned randomly to a buprenorphine dose-taper combined with either behavioral or standard treatment. Behavioral treatment included (a) a voucher incentive program for providing opioid-free urine samples and engaging in verifiable therapeutic activities and (b) the community reinforcement approach, a multicomponent behavioral treatment. Standard treatment included lifestyle counseling. Fifty-three percent of the patients receiving behavioral treatment completed treatment, versus 20% receiving standard treatment. The percentage of patients achieving 4, 8, 12, and 16 weeks of continuous opioid abstinence were 68, 47, 26, and 11 for the behavioral group and 55, 15, 5, and 0 for the standard group, respectively. Behavioral treatment improved outcomes during outpatient detoxification.

Allergen source materials and quality control of allergenic extracts.

Allergen extracts are prepared from a wide variety of source materials including pollens, fungi, arthropods, animal danders, foods, and dusts. The composition of allergen extracts can vary depending on the allergen source, manufacturing process, and storage conditions. Allergen-specific immunoglobulin E (IgE) assays and skin tests employ a variety of allergen-containing reagents that confer specificity on the test. Given that the allergen source materials are heterogenous mixtures of proteins, glycoproteins, carbohydrates, and other substances that are not allergenic, it is not unexpected that variability exists between test results obtained with different allergen-containing reagents. Variability within a single manufacturer's allergen product can be controlled by using reproducible extraction and processing procedures, single large lots of allergen source materials, and solid-phase supports. These controls do not, however, ensure the consistency of products between manufacturers or laboratories because allergen source materials, manufacturing procedures, and acceptance criteria for allergen reagents may vary.

Cooperative binding interactions required for function of the Ty1 sterile responsive element.

The Ste12p transcription factor controls the expression of Ty1 transposable element insertion mutations and genes whose products are required for mating in Saccharomyces cerevisiae. The binding site for Ste12p is a consensus DNA sequence known as a pheromone response element (PRE). Upstream activating sequences (UASs) derived from known Ste12p-dependent genes have previously been characterized to require either multiple PREs or a single PRE coupled to a binding site for a second protein. The Ste12p-dependent UAS from Ty1, called a sterile response element (SRE), is of the second type and is comprised of a PRE and an adjacent TEA (TEF-1, Tec1, and AbaA motif) DNA consensus sequence (TCS). In this report, we show by UV cross-linking analysis that two proteins, Ste12p and a protein with an apparent size of 72 kDa, directly contact the Ty1 SRE. Other experiments show that Tec1p is required for formation of the Ty1 SRE protein-DNA complex and is physically present in the complex. These results establish a direct role for Tec1p in the Ty1 SRE and yet another set of combinatorial interactions that achieve a qualitatively distinct mode of transcriptional regulation with Ste12p.

Oral immunotherapy with short ragweed extract in a novel encapsulated preparation: a double-blind study.

BACKGROUND: In the past, oral immunotherapy with allergens has had limited clinical effectiveness, presumably because of gastrointestinal destruction of allergens. OBJECTIVE: We have developed a new technique for microencapsulating protein antigens that permits them, when given orally, to bypass the stomach and be delivered to the small intestine in a highly immunogenic form. This study's purpose was to confirm the immunologic potency of orally administered short ragweed pollen extracts (SRW) microencapsulated (mSRW) by this new technique and to study the effectiveness of mSRW in controlling the symptoms of ragweed-induced hay fever. METHODS: Twenty-one SRW-sensitive patients were treated with mSRW in a double-blind placebo-controlled study. Serum SRW IgG and IgE antibodies and nasal secretory IgA antibodies were determined. During the ragweed season, symptoms were quantified by symptom-medication scoring. RESULTS: The treated patients had high titers of serum SRW IgG antibodies (1.15 microg/ml at baseline, increasing to 21.21 microg/ml), experienced regulation of the seasonal increase in serum SRW IgE antibodies (+9% vs +59% in placebo-treated patients), and produced a small amount of nasal SRW IgA antibodies. Despite an insubstantial pollen count, the symptom-medication scores in the treated group were lower than those in the placebo group (4.28 vs 6.18, p = 0.059), but the differences were statistically significant only in the subgroup that tolerated high doses (>20 microg of Amb a 1 in 19 of 21 patients, p = 0.04). These effects were accomplished without inducing any systemic reactions with a dose of mSRW (mean, 23.8 microg of Amb a 1) only slightly higher than that used in high-dose subcutaneous immunotherapy. CONCLUSION: Oral mSRW seems a safe, easily administered, and immunologically potent treatment for ragweed-induced hay fever, but its ultimate utility requires further study.

Intradermal reactivity to various insect and arachnid allergens among dogs from the southeastern United States.

OBJECTIVE: To compare cutaneous reactivity to insect and arachnid allergens in clinically normal (control) and allergic dogs in the southeastern United States. DESIGN: Prospective, controlled study. ANIMALS: 26 clinically normal dogs and 82 allergic dogs from the southeastern United States. PROCEDURE: Intradermal skin testing with various dilutions of 13 insect and arachnid allergens was performed on control dogs to establish skin threshold concentrations (ie, concentrations to which < 25% of the dogs had positive reactions). These established threshold concentrations were then used to test allergic dogs for reactivity. Prevalence of single and multiple insect and arachnid reactions were determined. RESULTS: Flea allergen was the only allergen that caused a significantly higher prevalence of positive reactions in allergic dogs than in control dogs. CLINICAL IMPLICATIONS: Flea hypersensitivity is the most important arthropod hypersensitivity in dogs. The importance of reactivity to insect and arachnid allergens other than flea allergen can be determined only when prevalence of positive reactivity has been determined in an appropriate regional control group of dogs.

Effects of mold proteases on the biological activity of allergenic pollen extracts.

OBJECTIVE: To determine the effect of mold extracts with high protease activity on the biological activity of allergenic tree, grass, and weed extracts, using in vivo and in vitro methods, in atopic dogs. ANIMALS: 15 dogs with history and clinical signs of atopy. All dogs had strong positive reactions (3+ or 4+) to 1 or more preselected allergens and negative reactions (O) to molds. PROCEDURE: Mold extracts and saline solution were coincubated separately with tree, grass, and weed pollen extracts at 4 C for 30 and 80 days. Skin end-point titration (30-day incubation) and ELISA inhibition (30- and 180-day incubations) tests were performed on all samples. The biological activity of pollen extracts coincubated with mold extracts was compared with that of pollen extracts coincubated with saline solution. RESULTS: In the skin end-point titration test, weed pollen extracts coincubated with a mixed mold extract lost a statistically significant amount of biological activity, compared with saline coincubated controls. In the ELISA inhibition test, grass and weed pollen extracts incubated with a mixed mold extract lost a significant amount of biological activity, compared with saline coincubated controls. A significant correlation in the measurement of biological activity was found between a loss of end-point dilution in the skin end-point titration test and a decrease in relative potency, as measured by the ELISA inhibition test for allergenic grass and weed extracts. CONCLUSION AND CLINICAL RELEVANCE: Mold proteases can decrease the biological activity of certain grass and weed pollen extracts when coincubated in the same vial for 30 days. Separation of mold and pollen extracts, when preparing immunotherapy vaccines, may help prevent loss of pollen extract potency and increase the vaccine's stability and efficacy.