Bone marrow storage and delayed consumption at Middle Pleistocene Qesem Cave, Israel (420 to 200 ka).

Bone marrow and grease constitute an important source of nutrition and have attracted the attention of human groups since prehistoric times. Marrow consumption has been linked to immediate consumption following the procurement and removal of soft tissues. Here, we present the earliest evidence for storage and delayed consumption of bone marrow at Qesem Cave, Israel (~420 to 200 ka). By using experimental series controlling exposure time and environmental parameters, combined with chemical analyses, we evaluated bone marrow preservation. The combination of archaeological and experimental results allowed us to isolate specific marks linked to dry skin removal and determine a low rate of marrow fat degradation of up to 9 weeks of exposure. This is the earliest evidence of such previously unidentified behavior, and it offers insights into the socio-economy of the human groups who lived at Qesem and may mark a threshold to new modes of Palaeolithic human adaptation.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

ABO incompatibility does not influence transfusion requirements in patients undergoing single-unit umbilical cord blood transplantation.

Hematopoietic stem cell transplantation is usually performed without considering the ABO compatibility between donor and recipient. There are few studies analyzing ABO matching impact on transfusion outcome of umbilical cord blood transplantation (UCBT) recipients. The aim of this study was to analyze factors influencing transfusion outcome, highlighting the ABO matching between donor and recipient. This study has reviewed data from 318 patients who underwent single unit UCBT at la Fe University Hospital from January 2000 to December 2014. There were no differences between RBC and platelet (PLT) requirements or RBC and PLT transfusion independence according to ABO matching between donor and recipient. RBC and PLT requirements were statistically correlated (ρ=0,841, P<0.001). A total of 170 and 188 patients achieved RBC and PLT independence, respectively, within 180 days after UCBT. Persistence of recipient isoagglutinins was detected in 6.8% of patients with major ABO incompatibility at median of 176 days (103-269) after UCBT. Autoimmune haemolytic anemia was diagnosed in 15 patients, 12 of them due to cold antibodies. In conclusion, ABO matching has not influenced transfusion requirements of patients undergoing UCBT.

Real-life management of primary immune thrombocytopenia (ITP) in adult patients and adherence to practice guidelines.

Very few data exist on the management of adult patients diagnosed with primary immune thrombocytopenia (ITP). The objectives of this study were to describe the diagnostic and treatment patterns for ITP and to compare the findings to recent ITP guidelines. We retrospectively analyzed the medical records of adult ITP patients diagnosed with primary ITP between January 2011 and June 2012 and examined whether management strategies were consistent or not with eight recent guideline-recommended practices. Overall, median age at the diagnosis of the disease (n = 101) was 58 years and median platelet count 12 × 10(9)/L with 75.2 % of patients having symptoms of ITP. The study perceived two major shortcomings in the diagnostic approach: (1) failure to perform peripheral blood film examination in 22.8 % of patients, a test that is mandatory by all guidelines, and (2) ordinary bone marrow assessment in more than half of the patients at diagnosis (50.5 %), a test not routinely recommended by guidelines. Low appropriateness in therapeutic management of patients included (1) unjustified use of intravenous immunoglobulin in the absence of bleeding in 54.8 % of patients and (2) splenectomy not being deferred until 6-12 months from diagnosis (median 161 days). Data also reflect a trend towards the early use of thrombopoietin receptor agonists in the treatment of patients who are refractory to any first-line therapy. We have recognized important areas of inapropriateness in the diagnostic and therapeutic management of adult ITP patients. Compliance with established guidelines should be encouraged in order to improve patient outcomes.

Incidence and risk factors of post-engraftment invasive fungal disease in adult allogeneic hematopoietic stem cell transplant recipients receiving oral azoles prophylaxis.

Studies that analyze the epidemiology and risk factors for invasive fungal disease (IFD) after engraftment in alloSCT are few in number. This single-center retrospective study included 404 alloSCT adult recipients surviving >40 days who engrafted and were discharged without prior IFD. All patients who received ⩾20 mg/day of prednisone were assigned to primary oral prophylaxis (itraconazole or low-dose voriconazole). The primary end point was the cumulative incidence (CI) of probable/proven IFD using the European Organization for Research and Treatment of Cancer and Mycoses Study Group (EORTC/MSG) criteria. The independent prognostic factors after multivariate analyses were used to construct a post-engraftment IFD risk score. The 1-year CI of IFD was 11%. The non-relapse mortality was 40% in those developing IFD and 16% in those who did not. The intent-to-treat analysis showed that 17% of patients abandoned the assigned prophylaxis. Age >40 years, ⩾1 previous SCT, pre-engraftment neutropenia >15 days, extensive chronic GVHD and CMV reactivation were independent risk factors. The post-engraftment IFD score stratified patients into low risk (0-1 factor, CI 0.7%), intermediate risk (2 factors, CI 9.9%) and high risk (3-5 factors, CI 24.7%) (P<0.0001). The antifungal prophylaxis strategy failed to prevent post-engraftment IFD in 11% of alloSCT. Our risk score could be useful to implement risk-adapted strategies using antifungal prophylaxis after engraftment.

A polymorphism in the XPD gene predisposes to leukemic transformation and new nonmyeloid malignancies in essential thrombocythemia and polycythemia vera.

Patients with essential thrombocythemia (ET) and polycythemia vera (PV) have an increased incidence of acute myeloid leukemia and new nonhematologic malignancies compared with the general population. However, information on the factors determining the risk for such complications is limited. In the present study, we investigated whether constitutional genetic variations in DNA repair predispose to leukemic transformation and new nonmyeloid neoplasias in patients with ET and PV. Case-control studies for predisposition to both types of malignancies were nested in a cohort of 422 subjects diagnosed with ET or PV during the period 1973-2010 in several institutions in Spain. A total of 64 incidence cases of leukemia and 50 cases of primary nonmyeloid cancers were accrued. At conditional regression analysis, the Gln/Gln genotype in the XPD codon 751 showed the strongest association with both leukemic transformation (odds ratio [OR] = 4.9; 95% confidence interval [95% CI], 2.0-12) and development of nonmyeloid malignancies (OR = 4.2; 95% CI, 1.5-12). Additional predictive factors were exposure to cytoreductive agents for leukemic transformation (OR = 3.5; 95% CI, 2.0-6.2) and age for nonmyeloid malignancies (OR = 2.0; 95% CI, 1.4-2.8). These findings provide further evidence about the contribution of inherited genetic variations to the pathogenesis and clinical course of myeloproliferative neoplasms.

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.

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.

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.

Genetically engineered hybrid proteins from Parietaria judaica pollen for allergen-specific immunotherapy.

BACKGROUND: Despite the use of conventional allergen-specific immunotherapy in clinical practice, more defined, efficient, and safer allergy vaccines are required. OBJECTIVE: The aim of the study was to obtain hypoallergenic molecules by deleting B-cell epitopes, which could potentially be applied to Parietaria judaica pollen allergy treatment. METHODS: Three hybrid molecules (Q1, Q2, and Q3) derived from fragments of the 2 major P judaica pollen allergens, Par j 1 and Par j 2, were engineered by means of PCR. Hybrid structures were compared with their natural components by means of circular dichroism, and their biologic activities were compared by using T-cell proliferation assays. Their IgE-binding activity was determined with Western blotting, skin prick tests, and enzyme allergosorbent and ELISA inhibition tests. RESULTS: The hybrid proteins, especially Q2 and Q3, revealed significantly reduced IgE reactivity compared with the natural allergens, as well as with the whole P judaica extract. Furthermore, in vivo skin prick tests showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the whole P judaica extract. Two (Q1 and Q2) of the 3 hybrid proteins induced a comparable T-cell proliferation response as that produced by the whole extract and natural allergens. CONCLUSION: Considering its reduced anaphylactogenic potential, together with its conserved T-cell reactivity, the engineered Q2 protein could be used in safe and shortened schedules of allergen-specific immunotherapy against P judaica pollen allergy. CLINICAL IMPLICATIONS: Recombinant hybrid Q2 is able to induce T-cell proliferation, thus evidencing a potential therapeutic effect. Its reduced IgE-binding capacity envisages an excellent safety profile.

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.

Tolerance and immunological changes of chemically modified allergen vaccine of Parietaria judaica in accelerated schedules.

The physicochemical modification of allergen vaccines provides a chance for administering higher doses in a shorter period of time. We sought to assess the safety and immunological changes of using a biologically standardized and modified Parietaria judaica pollen extract in accelerated schedules. Two accelerated schedules were tested in 45 P. judaica-allergic patients: 20 patients reached the maximum dose after two visits using two different concentrations and 25 patients reached the maximum dose after only one visit with two injections of the maximum concentration vial. The tolerance was assessed by recording all side effects related with immunotherapy. Specific antibody levels against native extract and rPar j 2 allergen were evaluated at the beginning and the end of the study. Allergenic potency determined by enzyme allergosorbent test (EAST) inhibition and skin prick test showed that modified P. judaica pollen had a 99.9% less allergenicity than native extract. After 650 doses administered, two clinically irrelevant local reactions (diameter<0 x 5 cm) and no systemic reactions were registered. Significant increases in allergen-specific IgG4 and IgG against P. judaica extract and rPar j 2 and significant decrease of specific IgE against Par j 2 were observed. The modified extract of P. judaica is safe to treat sensitive patients, even at accelerated regimens, and induces significant immunological changes.

Diagnosis of Parietaria judaica pollen allergy using natural and recombinant Par j 1 and Par j 2 allergens.

BACKGROUND: Parietaria judaica pollen is one of the main causes of allergic diseases in the Mediterranean area and contains two major allergens, called Par j 1 and Par j 2. OBJECTIVE: To evaluate the diagnostic potential of natural and recombinant forms of Par j 1 and Par j 2 in comparison with standardized P. judaica pollen extract. METHODS: Thirty patients allergic to P. judaica pollen and 15 control patients were investigated. Skin prick tests and determination of specific IgE levels were performed with commercial P. judaica extract, natural Par j 1 and Par j 2, and recombinant forms of both allergens expressed in P. pastoris. RESULTS: The whole group of patients with allergy to P. judaica had a positive skin test reaction to purified nPar j 1-Par j 2 and rPar j 2 at 5 microg/mL, and no false-positive reactions were detected. Natural and recombinant Par j 1 and Par j 2 showed no significantly different responses in skin tests compared with P. judaica extract. A high correlation was found between the serum-specific IgE levels to P. judaica extract vs. natural (R=0.996; P<0.001) and recombinant allergens (R=0.887 and 0.982 for rPar j 1 and rPar j 2, respectively; P<0.001). rPar j 2 displayed a 100% sensitivity and specificity among P. judaica-allergic patients. CONCLUSIONS: In vivo and in vitro diagnosis of P. judaica pollen allergy could be simplified using rPar j 2. This protein showed comparable IgE response and skin prick reactivity with those produced by P. judaica pollen extract.

Correlation between Olea europaea and Parietaria judaica pollen counts and quantification of their major allergens Ole e 1 and Par j 1-Par j 2.

BACKGROUND: In patients with pollinosis, allergic symptoms are often correlated with the number of airborne pollen grains, although this correlation is not always close. The direct measurement of the concentration of aeroallergens has only recently been introduced and is an important advance in public health information systems. OBJECTIVE: To compare specific quantification of aeroallergens Ole e 1 and Par j 1-Par j 2 Olea and Urticaceae pollen counts. METHODS: The Hirst method sampler and the Burkard Cyclone sampler were used for pollen count and allergen quantification, respectively. The aerosol was extracted and quantified for Ole e 1 and Par j 1-Par j 2 content using enzyme-linked immunosorbent assay procedures. RESULTS: Day-to-day variations were observed in both the pollen count and the amount of allergens. Pollen counts and aeroallergen quantification were closely correlated with 99% significance (Olea/Ole e 1: R = 0.892, P < .001; Urticaceae/Par j 1-Par j 2: R = 0.734, P < .001). CONCLUSION: The technique for the sampling and quantification of aeroallergens presented in this article, based on enzyme-linked immunosorbent assay and applied to the protein extracts directly obtained from the bioaerosol, represents an important advance in the epidemiologic study of allergic respiratory diseases.

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2.

BACKGROUND: Parietaria pollen is one of the most important causes of pollinosis in Mediterranean countries. Parietaria judaica pollen extract presents two major allergens, Par j 1 and Par j 2, that belong to the lipid transfer protein family. OBJECTIVE: To develop an ELISA for quantification of both major allergens of P. judaica pollen extracts, and to assert correlation of these allergens content with the allergenic activity of extracts. METHODS: Natural Par j 1-Par j 2 allergens were purified by gel filtration, ion exchange, and affinity chromatography and identified by mass spectrometry. Rabbit antisera were obtained using this protein preparation as antigen and used for immunoaffinity purification of nPar j 1-Par j 2. BALB/c mice were immunized with the immunopurified nPar j 1-Par j 2 and after fusion and screening by direct ELISA, 5D4 monoclonal antibody was selected as capture antibody to develop a quantitative two-site ELISA. Bound proteins were detected by a biotinylated Par j 1-Par j 2-specific polyclonal antibody. RESULTS: The optimized ELISA was developed from 25 to 8000 pg/mL of purified Par j 1-Par j 2, and a linear portion of 200-1000 pg/mL. The intraassay and interassay coefficients of variation were lower than 7% and 14% respectively. The assay was very sensitive and specific as it had a detection limit of 25 pg/mL and did not detect reactivity with the same family plants, as Urtica. Par j 1-Par j 2 allergens content was measured in 14 P. judaica and two P. officinalis pollen extracts showing a significant correlation with their allergenic activity measured by enzyme allergosorbent test inhibition. CONCLUSIONS: The results proved the usefulness of the two-sandwich ELISA for the standardization of Parietaria pollen extracts intended for clinical use, because of its good correlation with allergenic potency.

Development of a sandwich-type ELISA for measuring Pla a 1, the major allergen of Platanus acerifolia pollen.

BACKGROUND: Platanus acerifolia is an important cause of pollinosis in Western European cities. Pla a 1, a nonglycosylated 18-kDa protein with a prevalence of 80%, is a major allergen in P. acerifolia pollen extracts. Our aim was to develop a Pla a 1-specific ELISA to quantify this protein in allergenic extracts and preparations for clinical use. METHODS: Pla a 1 was purified by cation exchange at pH 7.0, gel filtration, and anion exchange chromatography at pH 10.0. Monoclonal (mAb) and polyclonal antibodies were obtained by immunizing mice and rabbits with nPla a 1. One (5C1) of the 13 mAb obtained was used as capture antibody at 5 mug/ml and biotin-labeled specific polyclonal antiserum at 0.63 microg/ml served for detection. RESULTS: The prevalence of Pla a 1-specific IgE to purified Pla a 1 among 47 P. acerifolia-allergic patients was 79%. The Pla a 1-ELISA developed has a linear range of 3-25 ng/ml, high sensitivity with a detection limit of 0.5 ng/ml and is highly specific as none of the 24 pollen, mite, mold, and plant food extracts tested gave positive results. The assay could quantify Pla a 1-like proteins in other planetree pollen extracts. A good correlation was obtained between Pla a 1 content of 11 P. acerifolia pollen extracts (average content 0.69% of the total protein) and their IgE-binding activity. CONCLUSIONS: The described two-site sandwich ELISA to measure Pla a 1 is useful for standardization of planetree pollen extracts intended for clinical use.

Pho d 2, a major allergen from date palm pollen, is a profilin: cloning, sequencing, and immunoglobulin E cross-reactivity with other profilins.

BACKGROUND: Up to now, some date palm pollen (DPP) allergens have been described but very few data are available about their molecular nature. The aim of this study was to identify and characterize Pho d 2, a major allergen from this pollen. METHODS: Sera from 25 patients allergic to DPP were analysed by immunoblotting. Purification of DPP profilin was performed by poly-l-proline affinity chromatography. Profilin-encoding cDNA from DPP was cloned by using a RT-PCR strategy and recombinant allergen was expressed as a non-fusion protein in Escherichia coli. Natural and recombinant Pho d 2 were investigated by means of enzyme allergosorbent test to compare the immunologic properties of both allergens and to analyse cross-reactivity with other profilins. RESULTS: A 14.4 kDa protein was identified as a major allergen in DPP extract. Purification, cloning, heterologous expression, and inhibition experiments identified it as profilin (Pho d 2). Pho d 2 comprises 131 amino acids and has high sequence identity with other allergenic food and pollen profilins. The prevalence of specific IgE antibody reactivity to natural Pho d 2 by ELISA was 56% and 64% by skin prick test (SPT). Pho d 2 is an important allergen as it is responsible for more than 70% of the IgE reactivity to the pollen extract. IgE directed against Pho d 2 showed a strong cross-reactivity with other profilins such as those from olive tree and grass pollens. CONCLUSION: Pho d 2, a 14.4 kDa protein identified as profilin, is a major and relevant allergen in DPP, as confirmed by SPT and thereby may elicit clinical symptoms in sensitized patients.

Quantification assay for the major allergen of Cupressus sempervirens pollen, Cup s 1, by sandwich ELISA.

BACKGROUND: The Cupressaceae are an important cause of pollinosis, particularly in Mediterranean countries. Cypress pollen allergenic extracts are difficult to produce since they have a low protein and a high carbohydrate content and consequently accurate standardization of these extracts is essential for diagnosis and immunotherapy. METHOD: Natural Cup s 1 was purified by a combination of hydrophobic interaction, gel filtration and ion exchange chromatographies and its enzymatic activity was analyzed. The allergen was used as reference material in the ELISA standard curve. The assay was based on a specific monoclonal antibody (3D2) immobilized on ELISA plates and used to capture Cup s 1. Bound proteins were detected by a combination of biotinylated specific antiserum and peroxidase-conjugated streptavidin. RESULTS: Purified Cup s 1 is a functional pectate lyase enzyme with a specific activity of 750 U/mg protein. The developed ELISA measured Cup s 1 concentrations ranging from 31.25 to 250 ng/ml in the lineal portion of the standard curve. The intra-assay and inter-assay variation coefficients in the working range were less than 8.1 % and 16 %, respectively. The assay was highly sensitive, with a detection limit of 3.8 ng/ml. The dose-response curves obtained with C. sempervirens pollen extracts and extracts belonging to other species from the Cupressaceae family showed a good parallelism compared with those obtained using the purified allergen, indicating that the same protein was measured. CONCLUSIONS: The assay described is sensitive, specific and reproducible for the quantification of Cup s 1 in C. sempervirens pollen extracts for clinical use. This ELISA could also be useful for other Cupressaceae-related pollen extracts.

Production profile of the major allergen Alt a 1 in Alternaria alternata cultures.

BACKGROUND: The fungus Alternaria is strongly associated with asthma, but the importance of fungal allergen products is frequently underestimated. The profile of allergen release from fungal material is poorly understood. OBJECTIVE: To investigate expression of the major allergen of Alternaria alternata, Alt a 1, during its growth in culture conditions for allergen extract production. METHODS: Allergen expression was examined by Alt a 1-specific 2-site monoclonal antibody enzyme-linked immunosorbent assay, immunoblotting, and potency assays. The release of Alt a 1 was studied by transmission electron microscopy in conjunction with immunogold staining by using antibodies with specificity for Alt a 1. RESULTS: A maximum amount of Alt a 1 was obtained after 4.5 weeks of growing, and it was found predominantly in the spent culture medium. In the same way, total IgE binding activity showed 15-fold more activity in the spent culture medium than in the buffer-extractable antigen fraction. Immunogold electron microscopy provided evidence that Alt a 1 is released from spores and mycelia. CONCLUSIONS: Alternaria alternata allergenic proteins were constantly released into the culture medium, where they accumulated. Alt a 1 was a good marker for checking optimal culture conditions for A alternata extract production intended for clinical use.