Utility of opium seed extract tests in preventing hypersensitivity reactions during surgery

BACKGROUND: Anaphylaxis during anaesthesia is fatal in 3-9% of patients and analgesics, including opioids, and is the second most common medicament-related cause, although the prevalence is underestimated. We recently found that patients may generate IgE antibodies to opium seeds. OBJECTIVES: To determine the diagnostic accuracy of specific antibodies to morphine, codeine, rocuronium and oil body and aqueous fractions of Papaver somniferum seeds in the diagnosis and prevention of allergy to opioids. METHODS: Patients with hypersensitivity reactions during surgery, and severe clinical allergy (pollen, tobacco), and illicit heroin users were selected. The sensitivity, specificity and predictive values of in vivo and in vitro diagnostic techniques including oil body and aqueous fractions of P. somniferum seeds were measured. RESULTS: We studied 203 patients, with mean age 35.1 ± 17.1 and 200 healthy controls. Patients sensitised to heroin or with hypersensitivity reactions during surgery responded to P. somniferum seed tests. Of patients not known to be sensitised to opioids, the highest positivity was in patients sensitised to tobacco (p < 0.001). Opium seed skin tests and IgE, especially the oil body fraction, were more sensitive (64.2%) and specific (98.4%) than morphine, codeine and rocuronium tests for opioid sensitivity. Pollen allergy was not a risk factor for sensitisation to morphine. CONCLUSIONS: Sensitivity to opioids and intraoperative anaphylaxis can be diagnosed by routine tests. IgE and skin tests for the oil body fraction of P. somniferum had the highest sensitivity for sensitisation to opioids

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In vitro and in vivo effects of neem tree (Azadirachta indica A. Juss) products on larvae of the sheep nose bot fly (Oestrus ovis L. Díptera: Oestridae).

Two studies were carried out in order to test the effects of neem tree extracts (Azadirachta indica A. Juss) on sheep bot fly larvae (Oestrus ovis L. Diptera: Oestridae). First, aqueous extracts from neem seeds (ASNE) at 0, 5 y 10% (w/v) concentrations were tested on larval mortality in vitro. In a second study, the effect of oral administration with neem seed meal (0, 100 y 200mg/kg) and neem leaves (1% of diet) on number of larvae found at necropsy and larval development was evaluated in experimentally O. ovis-infected sheep. Results in Experiment 1 showed a significant (P<0.05) effect of ASNE on time to L1 mortality in a dosis-dependent manner. In Experiment 2, oral administration of seeds or leaves did not affect the number of larvae found at necropsy of the sheep, but interfered with larval development and there was a tendency to reduce larval weight at the end of the infection period (55d).

Utility of opium seed extract tests in preventing hypersensitivity reactions during surgery.

BACKGROUND: Anaphylaxis during anaesthesia is fatal in 3-9% of patients and analgesics, including opioids, and is the second most common medicament-related cause, although the prevalence is underestimated. We recently found that patients may generate IgE antibodies to opium seeds. OBJECTIVES: To determine the diagnostic accuracy of specific antibodies to morphine, codeine, rocuronium and oil body and aqueous fractions of Papaver somniferum seeds in the diagnosis and prevention of allergy to opioids. METHODS: Patients with hypersensitivity reactions during surgery, and severe clinical allergy (pollen, tobacco), and illicit heroin users were selected. The sensitivity, specificity and predictive values of in vivo and in vitro diagnostic techniques including oil body and aqueous fractions of P. somniferum seeds were measured. RESULTS: We studied 203 patients, with mean age 35.1±17.1 and 200 healthy controls. Patients sensitised to heroin or with hypersensitivity reactions during surgery responded to P. somniferum seed tests. Of patients not known to be sensitised to opioids, the highest positivity was in patients sensitised to tobacco (p<0.001). Opium seed skin tests and IgE, especially the oil body fraction, were more sensitive (64.2%) and specific (98.4%) than morphine, codeine and rocuronium tests for opioid sensitivity. Pollen allergy was not a risk factor for sensitisation to morphine. CONCLUSIONS: Sensitivity to opioids and intraoperative anaphylaxis can be diagnosed by routine tests. IgE and skin tests for the oil body fraction of P. somniferum had the highest sensitivity for sensitisation to opioids.

Expression of endogenous retroviruses is negatively regulated by the pluripotency marker Rex1/Zfp42.

Rex1/Zfp42 is a Yy1-related zinc-finger protein whose expression is frequently used to identify pluripotent stem cells. We show that depletion of Rex1 levels notably affected self-renewal of mouse embryonic stem (ES) cells in clonal assays, in the absence of evident differences in expression of marker genes for pluripotency or differentiation. By contrast, marked differences in expression of several endogenous retroviral elements (ERVs) were evident upon Rex1 depletion. We demonstrate association of REX1 to specific elements in chromatin-immunoprecipitation assays, most strongly to muERV-L and to a lower extent to IAP and musD elements. Rex1 regulates muERV-L expression in vivo, as we show altered levels upon transient gain-and-loss of Rex1 function in pre-implantation embryos. We also find REX1 can associate with the lysine-demethylase LSD1/KDM1A, suggesting they act in concert. Similar to REX1 binding to retrotransposable elements (REs) in ES cells, we also detected binding of the REX1 related proteins YY1 and YY2 to REs, although the binding preferences of the two proteins were slightly different. Altogether, we show that Rex1 regulates ERV expression in mouse ES cells and during pre-implantation development and suggest that Rex1 and its relatives have evolved as regulators of endogenous retroviral transcription.

An observational study on outgrowing food allergy during non-birch pollen-specific, subcutaneous immunotherapy.

BACKGROUND: Birch pollen-specific immunotherapy (SIT) decreases allergy to foods containing birch pollen-homologous allergens. Cross-reactivity was also observed between plane tree pollen and some vegetable foods. OBJECTIVE: The aim of this study was to evaluate the outgrowing of food allergy by patients suffering from vegetable food allergy associated with plane tree pollinosis (rhinoconjunctivitis and/or asthma) during plane tree pollen SIT. METHODS: An observational and prospective study was conducted in 16 adult patients suffering from vegetable food allergy (hazelnut, walnut, lettuce, peach and cherry) and from plane tree pollinosis receiving plane tree pollen SIT for 1 year. Open oral challenges with the implicated food were performed before and after SIT. Blood samples were drawn for measurement of pollen- and food-specific IgE and IgG4 before and after treatment. RESULTS: Plane tree SIT resulted in a significant decrease in food allergy, since the mean food quantity provoking objective symptoms increased from 2.19 to 13.74 g (p < 0.05), and 6 of the 11 patients tolerated the highest level (25 g) of the challenged food after plane tree SIT. Laboratory data also showed a decrease in IgE levels and an increase in IgG4 levels after immunotherapy. CONCLUSION: SIT with plane tree pollen has a positive impact on food allergy in plane tree pollen-allergic subjects.

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.

Pollen allergy in peach-allergic patients: sensitization and cross-reactivity to taxonomically unrelated pollens.

BACKGROUND: Fruit allergy has been attributed to cross-reactive IgE to pollens and has been associated with a particular pollen sensitization. OBJECTIVE: The aim of the study was to evaluate sensitization to several taxonomically unrelated pollens in peach- and pollen-allergic patients and to study cross-reactivity between them. METHODS: One hundred sixty-five patients were evaluated: 70 peach- allergic patients together with 95 pollen-allergic patients (control group). Pollen skin tests in duplicate were performed to 5 grasses, 8 trees, and 7 weeds. Cross-reactivity between peach and taxonomically diverse pollens was determined by radioallergosorbent inhibition and Western blot inhibition tests. Experiments were also carried out after preadsorption of the sera with purified natural profilin. RESULTS: The skin test results revealed that peach-allergic patients frequently reacted to most pollens-grasses, weeds, and trees-even when some of these are not found in our geographic area. There was a statistically significant increase in sensitization frequency to most trees and weeds, with a statistically higher occurrence of asthma (odds ratio 2.98, 95% confidence interval 1.46-6.09). Inhibition test results provided evidence that taxonomically unrelated grasses, weeds, and trees produced various and substantial degrees of inhibition in specific IgE to peach and that the peach extract elicited strong inhibitions to those pollens. Profilin was found to be a relevant cross-reactive antigen in these patients. CONCLUSION: The results of this study provide evidence that peach allergy is linked to sensitization to several taxonomically unrelated pollens. This is attributable to the ubiquitous nature of the IgE binding determinants-such as profilins-between peach and taxonomically unrelated pollens.

Identification of a 36-kDa olive-pollen allergen by in vitro and in vivo studies.

BACKGROUND: Ole e 1 has been considered the major allergen of olive (Olea europaea) pollen. Some other relevant allergens (Ole e 2, 3, 4, and 6) have been recently described. This work aimed to study the IgE-binding frequency of a 36-kDa protein from O. europaea pollen in a large population of olive-allergic patients, its allergenic reactivity in vivo, and its presence in olive pollens of different origin, as well as in other relevant allergenic pollens. METHODS: Identification of IgE-binding components from O. europaea pollen extracts was elucidated by inhibition of SDS-PAGE immunoblotting using recombinant profilin (Ole e 2) and Ole e 1 molecules. The IgE-binding frequency of the 36-kDa protein was estimated by Western blot in a sample of 120 sera from olive-allergic patients. The cutaneous test with the 36-kDa protein was performed by intradermoreaction in allergic patients and control subjects. RESULTS: Exactly 83% of the sera from O. europaea-allergic patients recognized a protein with an apparent molecular weight of 36 kDa, under reducing conditions. It was detected by sera from monosensitized and polysensitized patients, showing a higher IgE frequency than the major allergen Ole e 1 (59%) and the minor profilin (Ole e 2) allergen (27%). Similar reactivity rates (79%) was found by intradermal test. Extracts from olive pollens collected in California presented a much higher amount (around 16-fold on average) of the 36-kDa protein than those from pollens of Spanish origin. The presence of similar allergens was detected only in closely related species (Syringa, Fraxinus, Ligustrum), and not in other common allergenic pollens. CONCLUSIONS: The 36-kDa protein constitutes a major allergen for olive-sensitized patients, but it is not equally represented in O. europaea pollens of different origins.

Occupational allergy caused by carnation (Dianthus caryophyllus).

BACKGROUND: Occupational respiratory symptoms caused by decorative flowers are seldom reported in the literature. In our area a large portion of the population works in carnation (Dianthus caryophyllus) winter quarters, and many workers have symptoms of rhinitis and asthma related to exposition. OBJECTIVE: The purposes of this study were to investigate whether the symptoms induced by carnation were IgE-mediated and to study the possible allergens involved. METHODS: A total of 16 subjects employed in indoor carnation cultivation with symptoms during exposition time were studied along with 15 patients with allergic asthma who were not exposed to carnations and 15 healthy carnation workers used as control subjects. Skin prick tests with carnation extract and RASTs were performed. Protein bands were isolated by SDS-PAGE, and afterwards immunoblotting was performed to characterize the extract. Specific nasal provocation and nonspecific bronchial provocation tests were performed for all the asthmatic patients. Diurnal variation in peak expiratory flow was also measured. RESULTS: Skin prick test responses with carnation extract were positive in 15 of the 16 patients and negative in all control subjects. Nasal provocation test responses with carnation extract were positive in 13 of 16 patients. A significant correlation was seen between RAST and nasal provocation results (P <.01). Immunoblotting of sera from 13 patients showed 2 major IgE-binding fractions of 34 and 35 kd in most of the patients, which could constitute the major allergen. Methacholine PD20 showed a variable degree of nonspecific bronchial hyperresponsiveness in all asthmatic subjects. CONCLUSION: Data demonstrate the involvement of carnation in occupational allergy, mediated by an IgE-dependent mechanism.

Rhinoconjunctivitis and asthma caused by vine pollen: a case report.

BACKGROUND: The vine (Vitis vinifera) is a cultivated plant that is found in some European and American countries. Its pollen gathers in small quantities during a short pollination period in the months of May and June. Allergy to vine pollen has not been previously documented. OBJECTIVE: We sought to describe a case report of allergy to vine pollen documented on the basis of anamnesis, cutaneous, provocation, and specific IgE determination tests. METHODS: An allergenic extract was obtained from collected V vinifera pollens by aqueous standard procedures. Pollen counts and pollination periods of this and other common pollens in the area where the patient became symptomatic were studied. Cutaneous tests and the presence of specific IgE to the pollen extracts were performed by prick, CAP, and RAST techniques. Bronchial and conjunctival tests with the involved pollen extracts were also carried out to identify the sensitizing allergens. Five healthy subjects and 5 pollinic patients were used as control subjects and underwent the same tests. RESULTS: Skin prick test responses with vine pollen at different concentrations were positive for the studied patient and negative for the control subjects. Patient serum revealed a total IgE titer of 334 IU/mL and a specific IgE value of 1.3 PRU/mL (RAST class 2) to vine pollen. Bronchial and conjunctival provocation test responses were also positive when the patient was challenged with V vinifera extract. CONCLUSION: Exposure to the pollen of the vineyard plants (V vinifera) can induce immunologic sensitization and rhinoconjunctivitis/asthma.

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.

Genes essential for nod factor production and nodulation are located on a symbiotic amplicon (AMPRtrCFN299pc60) in Rhizobium tropici.

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli. Here we report the isolation and molecular characterization of a symbiotic amplicon of Rhizobium tropici. To search for symbiotic amplicons, a cartridge containing a kanamycin resistance marker that responds to gene dosage and conditional origins of replication and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299. Derivatives harboring amplifications were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic amplicon, which we termed AMPRtrCFN299pc60, contains several nodulation and nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1. Amplification of AMPRtrCFN299pc60 through homologous recombination between ISRtr1 repeats increased the amount of Nod factors. Strikingly, the conjugal transfer of the amplicon into a plasmidless A. tumefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confined to an ampliable DNA region of the pSym.

Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin).

BACKGROUND: Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua. OBJECTIVES: We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy. METHODS: Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen. RESULTS: Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin. CONCLUSION: This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.

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.

Generation of Rhizobium strains with improved symbiotic properties by random DNA amplification (RDA)

To select for bacterial strains with enhanced phenotypes, random fragments of a whole genome, or a defined region of the genome, are cloned in a nonreplicating vector. The resulting plasmids are integrated by recombination into the homologous DNA region of the original strain. Integration gives rise to a nontandem direct duplication of the corresponding DNA region separated by the vector moiety of the plasmid. Recombination between the direct repeats leads to tandem duplication and further amplification of the entire integrated DNA, including the vector. Bacteria harboring the amplified DNA are selected by increasing the dosage of an antibiotic corresponding to a resistance marker of the integrated vector. Pooled strains carrying amplifications are then challenged with a selective pressure for the desired phenotype. After repeated selection cycles, the most fit strains are isolated. We used this process, which we called random DNA amplification, to select Rhizobium strains with increased competitiveness for nodule formation. Derivatives containing randomly amplified DNA regions of the symbiotic plasmid of Rhizobium tropici CFN299 strain were generated. Pools of amplified strains were inoculated onto various tropical legumes. After several cycles of selection through plants, amplified derivatives showing an increased competitiveness for nodule formation with the leguminous plant Macroptilium atropurpureum were obtained.

Production and characterization of profilin monoclonal antibodies.

Profilins have been identified as a pan-allergen of different plant pollens and foods. In this paper, we describe the generation of monoclonal antibodies (MAbs) by immunizing BALB/c mice with Helianthus annuus purified profilin in order to characterize this important and common allergen. A panel of forty MAbs has been obtained, and twenty of them were used to map antigenic determinants in this molecule. At least two different antigenic determinants were recognized in H. annuus profilin by immunoblotting. Using the purified MAbs produced against sunflower profilin, we have analyzed the common epitope determinants in pollens of different plants: Olea europaea, Cynodon dactylon, Mercurialis annua, Phleum pratense, Parietaria judaica and Betula verrucosa. These experiments showed different cross-reactivity patterns.

Recombinant DNA technology in allergology: cloning and expression of plant profilins.

Profilin, an ubiquitous protein involved in eukaryotic cytoskeleton regulation, has been previously described as allergen in grasses, weeds and trees and in many fruits and vegetables, and it is in part responsible for cross-reactivities pollen and food allergic patients. Completed cDNA clones coding for Phleum pratense, Olea europaea, Cynodon dactylon, Parietaria judaica and Helianthus annuus pollen profilins were isolated and sequenced. The deduced amino acid sequences share high identity with other plant profilins. Recombinant profilins were produced in Escherichia coli as non-fusion proteins. Induced cells produced high amounts of recombinant profilin, and after a single purification step on poly-(L-proline)-Sepharose, up to 45 mg of pure allergen per liter culture could be obtained. Recombinant profilins have similar allergenic determinants to their natural counterparts. The tertiary structure of Phleum pratense profilin described here showed three regions important for antibody recognition. The availability of a plant profilin tertiary structure opens future ways on the study of structure/antigenity relationships of these important allergens.

Mercurialis annua: characterization of main allergens and cross-reactivity with other species.

A multicentric study was conducted to evaluate the frequency of Mercurialis annua pollen sensitization in several areas of Spain and to select a population sample to characterize the main allergenic components in M. annua pollen. Patients were recruited from six hospitals in Spain. Out of 420 patients sensitized to pollens, 195 (46.4%) showed positive skin tests to M. annua, thus evidencing the high level of sensitization to the pollen of this plant in Spain. Thirty-seven sera with RAST class values to M. annua > or = 3 were selected for SDS-PAGE immunoblotting analysis. Two main allergenic components with molecular weights of 15.8 and 14.1 kD were detected in 59 and 51% of the sera, respectively, and they were identified as profilins. Isolation of the relevant allergens was made by affinity chromatography on a poly-L-proline-Sepharose column, followed by gel filtration and anion exchange chromatography in the micropreparative SMARTs System. A significant but low antigenic cross-reactivity between M. annua and Olea europaea, Fraxinus elatior, Ricinus communis, Salsola kali, Parietaria judaica and Artemisia vulgaris was demonstrated by several in vitro techniques.

IgE binding proteins in honey: discussion on their origin.

Pollen proteins, most frequently from Compositae plants, and glandular enzymes from Hymenoptera insects are the only honey allergens involved in every case of honey sensitization reported so far. Surprisingly, we found three patients, all showing the same pattern of honey sensitization with clinical history, cutaneous test and specific IgE clearly positive to honey crude extract but not to the aforementioned components. IgE binding bands, mainly at 54, 46, 17 and 16 kDa, were recognized by the sera of the three patients on blots following electrophoretic separation of a local honey extract under dissociating conditions. Attempts to identify these proteins with the most common honey allergens by means of SDS-PAGE immunoblotting failed, leaving open the question on the origin of protein allergens in cases of honey allergy. Thus, if bee-derived components are not proven to be involved in honey sensitization, other protein sources, such as nectar, should not be disregarded.