Clinical trial to assess tolerability and subrogate efficacy effects of an abbreviated schedule with house dust mites mixture subcutaneous immunotherapy.

Summary: Objective. To evaluate the tolerability and efficacy of Dermatophagoides pteronyssinus/Dermatophagoides farinae mixture subcutaneous immunotherapy (SCIT). Methods. Patients received an abbreviated build-up schedule. The aims were: number, percentage, and severity of adverse reactions. Secondary outcomes included: changes in immunoglobulin titers and changes in dose-response skin prick tests. Results. Out of 289 administrations, 17% elicited any clinically relevant adverse reaction. Most of them were local reactions (LR) (9.4%) and the rest (7.6%) were systemic. Significant increases in sIgG and sIgG4 were detected in serum samples. Cutaneous reactivity decreased significantly. Conclusions. SCIT with house dust mites mixture of ROXALL Medicina España S.A. seems to have an acceptable tolerability profile, induces blocking IgG and decreases skin reactivity.

Clinical assessment of tolerability, immunological and cutaneous reactivity effects of an abbreviated schedule with Olea europaeanative extract of subcutaneous immunotherapy.

Summary: Objectives. To evaluate the tolerability and efficacy of Olea europaea subcutaneous immunotherapy (SCIT) on patients with rhinoconjunctivitis. Methods. In this open clinical trial patients were assigned to an abbreviated build-up scheme. The outcomes were: number, percentage, and severity of adverse reactions. Secondary outcomes included: changes in immunoglobulin titers and changes in dose-response skin prick tests. Results. Only 8 systemic reactions were registered, which represented 7/47 (14.9%) of patients and 8/429 (1.9%) of administered doses. Regarding immunological parameters the significant increases of sIgG and sIgG4 evidenced the changes in the patient immune system. Cutaneous reactivity decreased significantly. Conclusions. Olea europaea SCIT (Allergovac® depot ROXALL Medicina España S.A.) showed a good safety and tolerability profile. Immunological changes with induction of blocking IgG and decreases in cutaneous reactivity were detected in the patients.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

Cupressus arizonica pollen: a new pollen involved in the lipid transfer protein syndrome?

BACKGROUND: Lipid transfer proteins (LTP) are responsible for systemic manifestations in food allergy. Their relationship with pollinosis is not clear. In our area, many patients allergic to multiple LTP-containing foods present pollinosis due to Cupressus arizonica. METHODS: We selected 6 patients with cypress pollinosis and food allergy to peach. Skin prick tests (SPT) were performed for pollens (grass, cypress, wall pellitory, plane tree, and olive tree) and plant foods (hazelnut, kiwifruit, peach peel, maize, wheat, peanut, lettuce, apple, mustard, and melon). In vitro assays included specific immunoglobulin (Ig) E to C arizonica and peach LTP (Pru p 3), enzyme allergosorbent test (EAST) inhibition, immunoblotting, immunoblotting-inhibition, and immunocytochemical techniques for the detection of Pru p 3-like LTP in cypress pollen grains. RESULTS: SPT were positive for C arizonica, peach, lettuce, mustard, and hazelnut in all patients. Specific IgE to C arizonica and Pru p 3 was positive in all but 1 patient, whose Pru p 3 IgE was negative. Immunoblotting under nonreducing conditions with C arizonica extract and patients' sera showed a band at 14-15 kDa that was inhibited by Pru p 3. Pru p 3 partially inhibited the C arizonica pollen extract in EAST-inhibition. Pru p 3-like LTP was localized in the cytoplasm and walls of C arizonica pollen grains. CONCLUSION: A 15-kDa allergen in C arizonica pollen was found in a group of patients presenting peach allergy and respiratory symptoms to cypress. In vitro tests and immunocytochemical techniques indicate that this protein is an LTP.

Neutrophil defensins: their possible role in allergic asthma.

BACKGROUND: Neutrophil defensins, originally identified as broad-spectrum antimicrobial peptides, have been implicated in the regulation of inflammatory and immunological processes. OBJECTIVES: To investigate whether the in vitro challenge of neutrophils from patients with bronchial asthma with allergens stimulated the release of alpha-defensins and whether levels released were dependent on lung infections. METHOD: The neutrophils were cultivated with different agonists and the concentration of alpha-defensin in cell-free supernatant was measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: Neutrophils from allergic patients released alpha-defensins via an allergen-dependent mechanism. Our results indicate that the in vitro activation of neutrophils is highly allergen-specific. In this context, allergens other than those which produced clinical symptoms did not elicit alpha-defensin release, and allergens had no effect on neutrophils from healthy donors. However, neutrophils from both allergic patients and healthy controls were able to release alpha-defensins upon treatment with PMA. In the allergen-stimulated neutrophils, cells from asthmatic patients stimulated with a sensitizing allergen showed a significantly higher production of alpha-defensin under respiratory tract infection than cells from the same patients without such an infection. CONCLUSION: Neutrophils from allergic patients release alpha-defensins via an allergen-dependent mechanism.

The Russian Thistle (Salsola kali) pollen major allergen, Sal k 1, can be quantified in allergenic extracts and airborne pollen.

BACKGROUND: Russian thistle (Salsola kali) pollen is an important cause of pollinosis in areas where rainfall is not abundant. Our aim was to develop an ELISA for quantification of the major allergen of S. kali extracts, Sal k 1, and to assess the correlation of this allergen content with the allergenic activity of extracts. METHODS: Sal k 1 was purified by ion exchange and gel permeation chromatography and identified by mass spectrometry. Monoclonal antibody 4C11 was used for capture at 5 microg/ml and biotin-labeled specific antiserum at 0.25 microg/ml served for detection. The allergenic activity of the pollen extracts was measured by enzyme allergosorbent test inhibition. RESULTS: Sal k 1 reacted to 85% of sera from 40 S. kali-allergic patients and was able to inhibit 92% of the IgE-binding capacity of patients' serum pool to the whole extract. The ELISA had a lineal range between 1.25 and 20 ng/ml of purified Sal k 1. The intra- and interassay coefficients of variation were lower than 5 and 10%, respectively. The assay was very sensitive since it had a detection limit of 0.08 ng/ml. No reactivity was found outside the Amaranthaceae family where only Kochia and Salicornia sp. gave significant reactivity. A good correlation (Spearman's rho = 0.92) was obtained between Sal k 1 content of different S. kali extracts and their IgE-binding activity. CONCLUSIONS: The results proved the usefulness of the two-site sandwich ELISA for aeroallergen control and for the standardization of S. kali pollen extracts intended for clinical use.

Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy.

BACKGROUND: Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT). OBJECTIVE: We sought to obtain hypoallergenic hybrid molecules which could potentially be applied to house dust mite (HDM) allergy treatment. METHODS: Two hybrid molecules (QM1 and QM2) derived from the two major Dermatophagoides pteronyssinus allergens, Der p 1 and Der p 2, were engineered by PCR, produced in Escherichia coli, and purified. The overall IgE-binding capacity of the hybrids was compared with their single components by Western blot, specific IgE, skin prick test (SPT), and IgE-inhibition assays. T cell proliferation assay were performed to confirm their retention of T cell reactivity. Immune responses to the hybrid molecules were studied in BALB/c mice. RESULTS: The IgE reactivity of both hybrid proteins was strongly reduced as evaluated by in vitro methods. Furthermore, in vivo SPTs performed on 106 HDM-allergic patients showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the individual components. Hybrid molecules induced higher T cell proliferation responses than those produced by an equimolecular mixture of Der p 1 and Der p 2. Immunization of mice with the hybrid proteins induced Der p 1- and Der p 2-specific IgG, which inhibited the binding of allergic patients' IgE to these natural allergens. CONCLUSION: QM1 and QM2 hybrids exhibited less IgE-binding activity but preserved immunogenicity and fulfilled the basic requirements for hypoallergenic molecules suitable for a future SIT of HDM allergy.

Guidelines on the clinical usefulness of determination of specific immunoglobulin E to foods.

The diagnostic gold standard for food allergy is challenge with the culprit food, particularly in double-blind placebo-controlled challenge. This approach involves risks and consumes both time and resources. A more efficient system would be desirable. The detection of serum specific immunoglobulin E (sIgE) against the culprit food enables us to establish sensitization, although this is not always accompanied by clinical reactivity. Age, symptoms (immediate/late reaction, local/systemic reaction), concomitant condition (eg, atopic dermatitis, pollinosis) and selection sample criteria (eg, presence of symptoms related to ingestion, positive skin prick test result) can influence the detection and concentration of IgE against foods. We analyze the clinical usefulness of sIgE determination in light of studies in which oral food challenge is used as the diagnostic method. We review clinical usefulness at diagnosis and in the decision to reintroduce the food, as well as the prognostic value of the determination of IgE to foods.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1.8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

Expression of a recombinant protein immunochemically equivalent to the major Anisakis simplex allergen Ani s 1.

BACKGROUND: Anisakis simplex is a nematode which can parasitize humans, producing anisakiasis and can induce immunoglobulin-(Ig)-E-mediated allergic symptoms. Parasite recombinant proteins, such as the major allergen Ani s 1, may be useful tools to avoid misdiagnosis of A simplex allergy due to cross-reactivity when whole parasite extracts are used. OBJECTIVE: To obtain Ani s 1 allergen as a recombinant protein with IgE-binding properties similar to its natural counterpart. METHODS: Ani s 1-encoding cDNA was amplified by polymerase chain reaction and cloned. The allergen was expressed in Escherichia coli as a nonfusion protein. Natural and recombinant Ani s 1 were investigated by means of Western blotting, enzyme allergosorbent test, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition using sera from 53 patients with A simplex allergy. RESULTS: Residues of the amino acid sequence of the encoded protein were 99.4% identical to the reported one. Purified rAni s 1 was obtained with a yield of 2 mg/L of culture while the yield of the natural counterpart was only 50 micro/g of larvae. rAni s 1 reactivity was not significantly different from that of the natural allergen; the correlation was excellent (p = 0.92, P < .001). ELISA-inhibition experiments showed that the dose-response inhibition curve obtained with rAni s 1 overlapped with that of nAni s 1. In an enzyme allergosorbent analysis, 86.8% of the A simplex-allergic patient sera reacted to rAni s 1. CONCLUSION: Recombinant Ani s 1 is immunochemically equivalent to its natural counterpart and therefore might be useful for the in vitro diagnosis of anisakiasis and A simplex-mediated allergy.

Cloning, expression and characterization of mugwort pollen allergen Art v 2, a pathogenesis-related protein from family group 1.

Mugwort (Artemisia vulgaris) belongs to the Compositae family, and is one of the main causes of allergy in late summer and autumn. The aim of the study was to characterize the allergen Art v 2 from mugwort pollen. Skin prick tests, performed in 19 patients allergic to mugwort and 10 control patients, showed an Art v 2 sensitization prevalence of 58%, whereas none false-positives were detected among control patients. Art v 2 was purified by standard chromatography and binding to Concanavalin A column and had an apparent molecular mass of 33 and 20 kDa, calculated by gel permeation and SDS-PAGE under denaturing conditions, respectively, showing that the allergen is composed of two identical subunits. Art v 2-encoding cDNA was amplified by PCR using degenerate primers based on reported partial amino acid sequences. Cloned cDNA encoding Art v 2 contains 140 bp that codify for a polypeptide of 15.8 kDa, with a predicted pI value of 5.2, and one potential N-glycosylation site. Protein homology search demonstrated that Art v 2 share 55-42% identical residues with pathogenesis-related protein PR-1 of tomato, potato, rape, wheat and rice. Homology was also found to Ves v 5 (41% identical residues). Bacterial-expressed recombinant Art v 2 was recognized only by 21% of mugwort-allergic patients. In conclusion, Art v 2 from mugwort is the first weed pollen allergen that belongs to the pathogenesis-related protein PR-1 and its recombinant form could help molecular diagnosis of mugwort associated allergy.

Biological standardization and maximum tolerated dose estimation of an Alternaria alternata allergenic extract.

BACKGROUND: The manufacture of allergenic extracts from the mold Alternaria alternata is influenced by factors such as strain variability, allergenic origin, culturing conditions and extraction process, which affect the reproducibility of the preparations intended for diagnostic and therapeutic use. OBJECTIVES: To select the most adequate antigenic source of A. alternata extracts and determine its maximum tolerated dose (MTD) to be used in a subsequent immunotherapy efficacy clinical trial. METHODS: Twenty-one patients monosensitized to A. alternata were involved in a biological standardization process of A. alternata extracts. Four different mold strains were cultured and used to produce extracts by three different methods, each incorporating proteins from different origins: culture filtrate, buffer extractable fraction and cellular antigens. The selected extract, characterized as in-house reference (IHR) preparation was used in a MTD finding immunotherapy study. Serum IgE, IgG, IgG1 and IgG4 specific of complete extract and purified natural and recombinant forms of Alt a 1 were determined by different EIA methods. RESULTS: Culture filtrate extract containing the allergens secreted to the spent medium was shown to be the most adequate option for establishing an IHR preparation for A. alternata extract manufacturing. A maximum dose of 1670 UBE, equivalent to 0.1 microg Alt a 1, was determined as MTD for immunotherapy. One year of administration of such a dose at monthly intervals elicited pronounced immunological changes with statistically significant decreases in IgE and increases in IgG4, both estimated with whole extract or purified Alt a 1. CONCLUSION: A high quality natural A. alternata extract has been developed and preliminarily tested to define its MTD for subsequent determination of the optimal dose in an immunotherapy efficacy clinical trial.

Sequence polymorphism and structural analysis of timothy grass pollen profilin allergen (Phl p 11).

Three cDNA clones encoding timothy grass pollen profilin (Phl p 11) were newly isolated. Comparison of the sequences of four cDNA clones, including a previously isolated clone, showed a low level of polymorphism. Isoelectrofocusing of highly purified timothy grass profilin indicated the existence of at least five isoforms. One recombinant profilin showed similar immunological properties to natural timothy grass profilin. Tertiary structure of Phleum pratense profilin was obtained by homology-based molecular modeling.

Sequencing and high level expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus.

The cDNA encoding an allergen from the dust mite Dermatophagoides pteronyssinus has been cloned and sequenced. The allergen (Der p 10) is a tropomyosin that shared more than 65% identical residues with other invertebrate tropomyosins. The final recovery of recombinant Der p 10 from the culture media after a single purification step was as much as 26 mg/l. The recombinant allergen is reactive to shrimp antitropomyosin IgG antibodies and has a 5.6% frequency of IgE reactivity in sera from mite-allergic patients.

Cloning and immunological characterization of the allergen Hel a 2 (profilin) from sunflower pollen.

Sunflower (Helianthus annuus) sensitization is not always related with occupational allergy. We have isolated the allergen profilin (Hel a 2) from this Compositae plant, cloned and sequenced five cDNAs encoding for full-length or partial Hel a 2. Natural sunflower profilin reacted with specific IgE in the 121 sera tested, at a frequency of 30.5%. Expression of the cDNA encoding Hel a 2 in Escherichia coli and a simple purification procedure by poly-L-proline chromatography allowed immunological characterization of the recombinant allergen. Binding of monoclonal antibodies against sunflower profilin revealed that some epitopes responsible for antigen-specific IgG production were not present in the recombinant allergen. High cross-reactivity has been found between recombinant Hel a 2 and profilins from other Compositae plants and also from botanically distant plants.

Phosphate control sequences involved in transcriptional regulation of antibiotic biosynthesis.

The expression of genes encoding enzymes involved in antibiotic and other secondary metabolite biosynthesis is down-regulated by easily assimilable phosphate, carbon and nitrogen sources. Phosphate control of antibiotic production appears to act at the transcriptional level by a mechanism similar to that involved in control of phosphatases and other phosphate-regulated enzymes. A phosphate control (PC) sequence, strikingly similar to the phosphate control (pho) boxes of many bacterial genes, has been isolated from the phosphate regulated promoter that controls biosynthesis of the antibiotic candicidin, and characterized. From computer analysis of sequence data, PC sequences appear to be associated with promoter regions of several phosphate-controlled antibiotic biosynthetic genes.

The evolutionary relationship of biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from gram-negative bacteria.

The Gram+ bacterium Rhodococcus globerulus P6 (RgP6) catabolizes a range of polychlorinated biphenyl (PCB) congeners, thus being of interest in bioelimination processes for PCB. The first step in the pathway, a dioxygenase attack of one of the biphenyl (BP) rings, is catalyzed by biphenyl dioxygenase (BDO). In this study, the nucleotide (nt) sequences of the four clustered cistrons, bphA1A2A3A4, encoding the subunits of BDO and forming part of the bph operon of RgP6 for BP degradation, were determined. A conserved motif proposed to bind a Rieske-type [2Fe-2S] cluster was identified in the deduced amino acid (aa) sequence of both the a subunit of the terminal oxygenase (BphA1) and ferredoxin (BphA3). The ferredoxin reductase subunit (BphA4) contains conserved sites for FAD and NADH binding. Deduced aa sequences of the BDO subunits shared homologies to multicomponent aromatic ring-hydroxylating dioxygenases from Gram- microorganisms. Stronger identity was found to toluene dioxygenase (TDO) of Pseudomonas putida F1 than to other BDO. Aa sequence comparisons suggest that BP degradation genes of RgP6 may have originated in Gram- microorganisms, probably Pseudomonas, and subsequently transferred to this Gram+ bacterium.

Phosphate control of pabS gene transcription during candicidin biosynthesis.

The pabS gene of Streptomyces griseus IMRU3570 encodes the enzyme p-aminobenzoic acid synthase, which synthesizes p-aminobenzoic acid (PABA), a precursor of the antibiotic candicidin (Cd). The pabS transcript reached a peak at 12 h of incubation in batch cultures, preceding the formation of PABA synthase and the antibiotic itself. A decay of the pabS transcript was observed with an apparent half-life of 35 min. Inorganic phosphate (Pi; 7.5 mM) reduced the synthesis of the pabS transcript by 90-95%, and consequently the formation of PABA synthase and Cd. Thirty min after addition of 7.5 mM Pi, the cells synthesized only about 15% as much pabS transcript compared to control cultures. However, Pi stimulated two- to threefold total RNA synthesis. The 1.7-kb pabS transcript shown by Northern hybridization was greatly reduced in amount in cells grown in 7.5 mM phosphate. Pi-deregulated mutants, described previously, were impaired in the transcriptional control exerted by Pi. It is concluded that Pi control of PABA synthase and Cd biosynthesis is exerted by repression of formation of the pabS mRNA.

Cloning and characterization of a phosphate-regulated promoter involved in phosphate control of candicidin biosynthesis.

Phosphate strongly repressed the formation of p-aminobenzoic acid (PABA) synthase, an enzyme involved in candicidin biosynthesis. Expression in Streptomyces lividans of the pabS gene (encoding PABA synthase) of Streptomyces griseus is repressed by phosphate at concentrations above 0.1 mM. However, expression of the pabS gene in Escherichia coli is not regulated by phosphate. Phosphate control of the expression of the pabS gene was observed in all plasmids containing the original 4.5-kb BamHI fragment, whereas no phosphate regulation was found when an upstream 1-kb fragment that carries the pabS promoter was deleted. Using the promoter-probe plasmid pIJ424, a '114-bp' promoter was cloned. Expression of the promoterless kanamycin phosphotransferase gene when fused to the '114-bp' promoter was strongly reduced by phosphate (90% at 5 mM concentration). The '114-bp' promoter has been sequenced and the first transcribed nucleotide identified by S1 mapping. The '114-bp' fragment is A + T-rich (54%), as compared to the Streptomyces genome (70-73% GC). The presence of a phosphate control sequence (pcs) in the upstream region of the pabS gene is proposed.

Quantification of profilins by a monoclonal antibody-based sandwich ELISA.

Profilins are plant allergens responsible for cross-reactivities in pollen and fruit-allergic patients. A two-site enzyme-linked immunosorbent assay has been developed for the quantification of profilins and its suitability for quantifying profilin in different plant extracts has been evaluated. The assay is based on two profilin-specific monoclonal antibodies (mAbs) with different epitope specificities. These antibodies were immobilized on ELISA plates and incubated with samples containing profilin. Bound profilin was detected by a combination of biotinylated profilin-specific antiserum and peroxidase-streptavidin conjugate. The optimized ELISA measured profilin concentrations ranging from 4 to 250 ng/ml and could quantify profilins from plant species of a variety of different botanical families. No reactivity to mites, molds, or crustaceans was detected, suggesting that the immunoassay is plant-specific. The results indicate that this sensitive profilin-assay will be helpful both for quantifying the profilin content of allergenic extracts intended for clinical use and for studying cross-reactivities between pollen extracts.