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An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen.

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.

Brassica napus↗

Silencing of the tobacco pollen pectin methylesterase NtPPME1 results in retarded in vivo pollen tube growth.

Sperm delivery in flowering plants requires extensive pollen tube growth through the female sporophytic tissues of the pistil. The apical cell wall emerges as a central player in the control of pollen tube growth, since it provides strength to withstand the internal turgor pressure, while imparting sufficient plasticity to allow cell wall extension through the incorporation of new membrane and wall material. Within this scenario, pectin methylesterases (PMEs; EC 3.1.1.11) emerge as crucial regulators in determining the mechanical properties of pectins, the major component of the apical pollen tube wall. We previously identified NtPPME1, a pollen specific PME from Nicotiana tabacum. Here we show that silencing of NtPPME1 results in a mild but significant decrease of in vivo pollen tube growth while the overall PME activity in pollen is not significantly affected. Although the precise mechanisms responsible for the observed phenotype are not known, it seems likely that the cell must maintain a closely regulated level of PME activity in order to maintain the equilibrium between strength and plasticity in the apical cell wall. A relatively minor disturbance of this equilibrium, as caused by NtPPME1 silencing, compromises pollen tube growth.

Carboxylic Ester Hydrolases↗

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth.

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H+ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 microM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H+ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H+ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen.

14-3-3 Proteins↗

Mitochondrial DNA decreases during pollen development in rapeseed (Brassica napus L.), but mitochondrial linear-plasmid-encoded RNA polymerase persists in mature pollen.

Mitochondrial DNA in the male reproductive cells of rapeseed (Brassica napus L.) was monitored by fluorescence microscopy of Technovit 7100 resin sections double-stained with 4',6-diamidino-2-phenylindole and 3,3'-dihexyloxacarbocyanine iodide. Mitochondrial DNA progressively decreased during pollen development and disappeared in mature pollen. This result corresponds well with the maternal inheritance of mitochondria in rapeseed determined by previous genetic analyses. To better characterize the mode of inheritance of the mitochondrial linear plasmid in rapeseed, which is transmitted through pollen, we analyzed by indirect immunofluorescence microscopy the expression and localization of ORF6 protein, a putative RNA polymerase encoded by the plasmid. ORF6 protein was expressed in mature pollen and specifically localized in the cytoplasm of sperm cells in the mature pollen. This suggests that the genes encoded by the plasmid DNA are transcribed in the mature pollen by its own RNA polymerase (ORF6 protein) and that the gene expression in the generative cells may be needed for transmission of plasmid DNA through the pollen.

Brassica napus↗

Comparison of recombinant timothy grass pollen allergens with natural extract for diagnosis of grass pollen allergy in different populations.

BACKGROUND: Complementary DNAs coding for the major timothy grass pollen (Phleum pratense) allergens Phl p 1, Phl p 2, and Phl p 5 and birch profilin were isolated, expressed as recombinant nonfusion proteins in Escherichia coli, and purified. OBJECTIVE: In this study the in vitro IgE-binding capacity of recombinant Phl p 1, Phl p 2, Phl p 5, and birch profilin and their IgE recognition frequencies were investigated by using sera from different populations. METHODS: One hundred eighty-three sera from patients allergic to grass pollen were obtained from different populations in Europe, Japan, and Canada. The sera were selected according to clinical criteria, skin testing, and RAST (CAP system; Pharmacia, Uppsala, Sweden) and then tested for IgE reactivity with natural and purified recombinant timothy grass pollen allergens by ELISA and Western blot. RESULTS: Most (94.5%) of the patients allergic to grass pollen could be diagnosed with a combination of recombinant Phl p 1, Phl p 2, Phl p 5, and profilin by means of ELISA. Sera that did not react with the recombinant allergens contained low levels of timothy grass pollen-specific IgE. Although considerable variability in IgE recognition frequency of the recombinant allergens was observed in certain populations, a good correlation was found between natural timothy CAP results and the combination of recombinant allergens in all 183 tested sera (r = 0.87). CONCLUSIONS: Despite considerable variability in the IgE recognition frequency, purified recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5) and profilin permitted successful in vitro diagnosis of grass pollen allergy in 94.5% of allergic individuals from different populations. The addition of other recombinant allergens (e.g., recombinant Phl p 4) would only slightly improve the in vitro test sensitivity.

Allergens↗

Human monoclonal IgG antibodies derived from a patient allergic to birch pollen as tools to study the in situ localization of the major birch pollen allergen, Bet v 1, by immunogold electron microscopy.

BACKGROUND: Bet v 1, the major birch pollen allergen, and related allergens present in various tree pollens, fruits, and vegetables represent a family of important cross-reactive allergens. Although the DNA, deduced amino-acid sequence, and structure of Bet v 1 have been determined, little is known regarding its biologic functions. OBJECTIVE: Human monoclonal antibodies derived from an individual allergic to birch pollen were used for refined ultrastructural localization of Bet v 1 in birch pollen grains to gain information regarding the allergen distribution and its possible biologic function. These data were to be supplemented by sequence analyses. METHODS: Ultrathin sections of anhydrously prepared birch pollen grains were incubated with human monoclonal antibodies (BAB1, BAB2, and BAB4). The binding sites were visualized in the transmission electron microscope by gold-conjugated anti-human IgG antibodies. RESULTS: In the cytoplasm of the birch pollen grain, human monoclonal antibodies bound to ribosome-rich areas and to pollen nuclei. Sequence analysis of Bet v 1 and homologous allergens identified a highly conserved p-loop motif in these proteins, which is typically found in nucleotide-binding proteins. CONCLUSIONS: Immunolocalization of Bet v 1 to ribosome-rich areas and the nucleus would be consistent with a highly conserved biologic function of Bet v 1 and homologous proteins as nucleotide binding proteins and explains why this group of plant proteins represents highly cross-reactive plant allergens against which many type I patients are sensitized.

Allergens↗

Pollen aperture heteromorphism. Variation in pollen-type proportions along altitudinal transects in Viola calcarata.

Some species produce pollen grains with different aperture numbers within a single individual (pollen aperture heteromorphism). In the pansy Viola diversifolia, aperture number is positively correlated with pollen germination speed, and negatively correlated with viability. In V. calcarata, young five-aperturate pollen grains germinate faster than four-aperturate ones. Heteromorphism could thus be favoured when pollination is unpredictable, as plants produce both very competitive and long-lived pollen grains. Depending on the efficiency of the pollinators, different proportions of pollen types will be optimal. In insect-pollinated species, such as V. calcarata, pollination efficiency generally decreases as elevation increases. We therefore expect a decrease in mean aperture number as altitude increases. This was found in four transects (out of six). Pollinator activity therefore has a potential impact on pollen morphology.

Plant Physiological Phenomena↗

Diagnosis of cypress pollen allergy: in vivo and in vitro standardization of a Juniperus ashei pollen extract.

BACKGROUND: Cypress pollen allergy is a major cause of rhinoconjunctivitis and asthma in the Mediterranean area. The nonstandardized cypress allergen extracts currently available for the diagnosis of cypress allergy have a low level of activity. The search for an active material has led to the selection of Juniperus ashei (Ja) pollen because of its very high cross-reactivity with cypress extracts and its superior allergenic activity. The aim of this study was to characterize in vitro and calibrate in vivo an in-house reference extract (IHRS) of J. ashei pollen and determine the specificity and sensitivity of a standardized Ja extract for the prick test diagnosis of cypress allergy. METHODS: Juniperus ashei pollen extract was analysed by 2-D electrophoresis. The IHRS Ja extract was calibrated by skin prick testing in 28 cypress-allergic patients. The sensitivity and specificity of cypress allergy diagnosis using a standardized Ja extract was studied by skin prick test in 42 cypress-allergic patients and 53 nonallergic patients. Jun a 1 content of the IHRS was determined by a monoclonal antibody-based electrophoretic technique. RESULTS: The Jun a 1 content of the 100 IR/ml Ja IHRS extract was 180 microg/ml. For in vivo diagnosis of cypress allergy, Ja pollen extract demonstrated a sensitivity of 95%, a specificity of 100%, a negative predictive value of 96%, and a positive predictive value of 100%. CONCLUSION: Standardized Ja pollen extract is therefore a very appropriate tool for the in vivo diagnosis of cypress pollen allergy and good candidate for specific immunotherapy.

Adolescent↗

How far can we simplify in vitro diagnostics for Fagales tree pollen allergy? A study with three whole pollen extracts and purified natural and recombinant allergens.

BACKGROUND: Current diagnostic tests for Fagales tree pollen allergy are often composed of mixtures of pollen of birch, alder and hazel. Their complex composition hampers accurate standardization. OBJECTIVE: The aim of this study was to investigate whether mixtures of tree pollen extracts can be replaced by a single pollen species, and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. METHODS: Sera (n = 1725) were selected on ground of a general suspicion for inhalant allergy, and tested in a RAST for birch, alder and hazel pollen. Sera with > 0.5 RU/mL for any of the three species were tested in a RAST for natural Bet v 1 and Bet v 2 as well as for recombinant versions of both allergens. RESULTS: Specific IgE antibodies (> 0.3 RU/mL) against birch, alder and hazel were found in 242, 298 and 292 sera, respectively. All sera with a positive RAST for alder and/or hazel and a negative RAST for birch were low-responder sera on alder and hazel, only five sera having a RAST value > 1.0 (all < 2.0). For all sera with a RAST > 0.5 RU/mL (n = 250), the mean of individual ratio's alder/birch and hazel/birch was 1.02 and 0.54, respectively. Of 223 of these sera, 63.2% had specific IgE against natural Bet v 1 and 63.7% against natural Bet v 2. When responses to both allergens were combined 93.7% were positive. The mean ratios Bet v 1 + 2/extract were 1.00, 1.04 and 2. 11 in case of birch, alder and hazel, respectively. For 211 sera the same analysis was performed with recombinant Bet v 1 and Bet v 2. Only six sera with Bet v 1-specific IgE (all < 0.5 RU/mL) were negative (< 0.3 RU/mL) on recombinant Bet v 1. For Bet v 2, 77/132 sera with specific IgE to the natural allergen did not react to the recombinant version. Twelve false-negatives had RAST values > 1.0 RU/mL. The mean of the individual recombinant/natural ratios was 0. 98 for Bet v 1 and 0.38 for Bet v 2 (P < 0.001). The mean ratio rBet v 1 + 2/birch was 0.75 with 17.5% false-negatives on the combination of recombinant allergens. CONCLUSION: Reliable in vitro diagnosis is possible with a single tree pollen extract (birch or alder). The same is true for purified natural Bet v 1 and Bet v 2. A combination of recombinant molecules is slightly less efficient.

Allergens↗

Soybean (Glycine max) pollen germination characteristics, flower and pollen morphology in response to enhanced ultraviolet-B radiation.

BACKGROUND AND AIMS: Ultraviolet-B (UV-B) radiation effect on reproductive parts of the plants has received little attention. We studied the influence of UV-B radiation on flower and pollen morphology, pollen production and in vitro pollen germination and tube growth of six genotypes of soybean (Glycine max). METHODS: Soybean genotypes were investigated by growing them under four levels of biologically effective UV-B radiation of 0 (control), 5, 10 and 15 kJ m(-2) d(-1) in sunlit controlled-environment chambers. KEY RESULTS: Reductions in lengths of flower, standard petal, and staminal column along with reduced pollen production, germination and tube growth were observed in all genotypes with increasing UV-B radiation. Combined response index (CRI), the sum of percentage relative responses in flower size, pollen production, pollen germination and tube growth due to UV-B radiation varied with UV-B dosage: -67 to -152 with 5 kJ m(-2) d(-1), -90 to -212 with 10 kJ m(-2) d(-1), and -118 to -248 with 15 kJ m(-2) d(-1) of UV-B compared to controls. Genotypes were classified based on the UV-B sensitivity index (USI) calculated as CRI per unit UV-B, where D 90-9216, DG 5630RR and D 88-5320 were classified as tolerant (USI > -7.43), and DP 4933RR, Stalwart III and PI 471938 were sensitive (USI < -7.43) in their response to UV-B radiation. Pollen grains produced in plants grown at 15 kJ m(-2) d(-1) UV-B radiation were shrivelled and lacked apertures compared to control and other UV-B treatments in both sensitive and tolerant genotypes, and the differences were more conspicuous in the sensitive genotype (PI 471938) than in the tolerant genotype (D 90-9216). The number of columellae heads of the exine was reduced with increasing UV-B radiation. CONCLUSIONS: Soybean genotypes varied in their reproductive response to UV-B radiation. The identified UV-B tolerant genotypes could be used in future breeding programmes.

Dose-Response Relationship, Radiation↗

Analysis of expressed sequence tags from Cryptomeria japonica pollen reveals novel pollen-specific transcripts.

Cryptomeria japonica D. Don is one of the most important forest trees in Japan, but more than 10% of the Japanese population is allergic to its pollen. We constructed a cDNA library derived from pollen grains of C. japonica and performed an analysis of expressed sequence tags (ESTs). We obtained partial sequences from 1929 clones, which represented 1365 unique transcripts. Among the unique transcripts, 984 (72%) encoded proteins that were similar to Arabidopsis proteins with E-values of < 10(-5). Analysis of funtional composition of the pollen ESTs revealed the overrepresentation of mRNAs for proteins involved in protein synthesis and post-translational modification. The most abundant transcripts were derived from novel genes (CjMP1-related genes) and encoded proteins that were not homologous to any proteins in current databases. The CjMP1-related genes formed a multi-gene family and were expressed specifically in the pollen grains of C. japonica. An analysis of homologies between ESTs from C. japonica pollen and proteins in the Structural Database of Allergenic Proteins revealed that products of 48 of the clones (2.5%) exhibited significant homology to known plant allergens. Our results provide new information about pollen-specific genes and potential allergens in C. japonica pollen.

Amino Acid Sequence↗

Hexose transport in growing petunia pollen tubes and characterization of a pollen-specific, putative monosaccharide transporter.

We investigated the molecular and physiological processes of sugar uptake and metabolism during pollen tube growth and plant fertilization. In vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only in medium containing sucrose (Suc) as a carbon source, but also in medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis demonstrated a rapid and complete conversion of Suc into equimolar amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petunia monosaccharide transporter 1), which is highly homologous to plant monosaccharide transporters, was isolated from petunia. Pmt1 belongs to a small gene family and is expressed specifically in the male gametophyte, but not in any other vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature and germinating pollen. A model describing the transport of sugars to the style, the conversion of Suc into Glc and Fru, and the active uptake by a monosaccharide transporter into the pollen tube is presented.

Amino Acid Sequence↗

Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2.

We screened for pollen-specific kinase genes, which are potential signal transduction components of pollen-pistil interactions, and isolated two structurally related receptor-like kinases (RLKs) from tomato, LePRK1 and LePRK2. These kinases are similar to a pollen-expressed RLK from petunia, but they are expressed later during pollen development than is the petunia RLK. The abundance of LePRK2 increases when pollen germinates, but LePRK1 remains constant. Both LePRK1 and LePRK2 are localized to the plasma membrane/cell wall of growing pollen tubes. Both kinase domains have kinase activity when expressed in Escherichia coli. In phosphorylation assays with pollen membrane preparations, LePRK2, but not LePRK1, is phosphorylated, and the addition of tomato style, but not leaf, extracts to these membrane preparations results at least partially in specific dephosphorylation of LePRK2. Taken together, these results suggest that LePRK1 and LePRK2 play different roles in postpollination events and that at least LePRK2 may mediate some pistil response.

Amino Acid Sequence↗

Effect of nedocromil sodium on histamine airway responsiveness in grass-pollen sensitive asthmatics during the pollen season.

We examined the effect of nedocromil sodium on histamine airway responsiveness in twelve grass-pollen sensitive patients during the 1984 pollen season. The study was a randomized double-blind crossover comparison of nedocromil sodium administered by a pressurized aerosol (4 mg b.d.) with placebo. Crossovers were made at 14-day intervals throughout 8 weeks of the grass pollen season. Histamine airway responsiveness was assessed twice before the pollen season and at the end of each 14-day treatment period. Results were expressed as the provocation concentration (PC) producing a 10% fall in FEV1 (PC10 FEV1) and a 40% fall in flow at 30% of the vital capacity (PC40 V30(P]. During the pollen season all patients developed hay fever and seven had symptoms of asthma. The observed lowest values of PC10 FEV1 and PC40 V30(P) during the placebo treatment periods were significantly lower than mean preseasonal values although not significantly lower than theoretical expected values. Geometric means PC10 FEV1 and PC40 V30(P) were significantly higher during nedocromil sodium treatment compared with placebo. These results indicate that nedocromil sodium has a small but statistically significant effect reducing histamine airway responsiveness in grass-pollen sensitive patients during the pollen season.

Adult↗

Tree pollen allergy. II. Sensitization to various tree pollen allergens in Sweden. A multi-centre study.

In a multi-centre study, comprising 16 clinics, 871 adult hay fever patients were studied. Most patients included had springtime hay fever. Skin prick tests were performed with 20 different tree or bush pollen (lignoses) allergens. RAST determinations were done with eight different tree pollen allergens in 590 patients. Birch pollen (BP) allergen gave the highest frequency of positive test results. The test results with the various tree pollen allergens in patients with BP allergy were compared with those in patients without BP allergy. With all the allergens a significantly higher frequency of positive test results was found in those with BP allergy than in those without. Only very small differences were noted between various parts of the country, and sensitization against various tree pollens was common even in regions where the corresponding trees do not grow. It was concluded that a high degree of cross-sensitization exists among tree pollens, and testing with BP allergen is sufficient for the diagnostic screening of tree pollen allergy.

Humans↗

Immunotherapy with partially purified and standardized tree pollen extracts. I. Clinical results from a three-year double-blind study of patients treated with pollen extracts either of birch or combinations of alder, birch and hazel.

Fifty-four adult patients with tree pollen-induced rhinitis (28), asthma (1), or rhinitis and asthma (25) were selected for immunotherapy with standardized and partly purified tree pollen extracts using a double blind protocol. The selection was based on clinical history, results of nasal or bronchial challenge, skin prick tests and RAST. Further, based on crossed radio-immunoelectrophoresis, sex, age and severity of symptoms, the patients were allocated in matched pairs and the treatment alternatives were randomly distributed within the pairs. Twenty-three patients treated with extracts composed of any combination of alder, birch and hazel pollen which matched their IgE response in CRIE (Group 1 (ABC)) and 22 patients receiving birch pollen extracts (Group 2 (B)) completed all 3 years of treatment. The in vivo results comprising symptom and medicine consumption scores are given here. Changes in specific skin and nasal reactivity as well as in immunological parameters are presented separately. No significant differences were demonstrated between the treatment groups in the two parameters. Both extracts were effective and reduced in general the symptom scores to one tenth of the starting level. Expressed another way, at the end of the study, the patients tolerated 30 times more pollen until symptoms of the same severity were elicited, compared to before. In the Nordic countries, spring-time asthma and rhino-conjunctivitis caused by pollen from deciduous trees can be effectively treated with an extract of birch pollen alone.

Adolescent↗

Quantification of pollen solute release using pollen grain column chromatography.

The impact of pollen on the respiratory mucosa was modeled by studying the process by which solutes are eluted from pollen grains. Rye grass (Lolium perenne), short ragweed (Ambrosia artemisiifolia), and white oak (Quercus alba) pollens were packed between glass wool plugs in small columns. Water was pumped through the columns and the eluate solute yield was determined by measurement of the dry solute weight. Solute separation was rapid, and concentrations and osmolalities of the eluate decreased exponentially. Theoretical initial solute concentrations were 179 g/l for rye grass, 55 g/l for short ragweed and 349 g/l for oak pollen eluates. Theoretical initial osmolarities of the same eluates were 321 mOsm/kg for rye grass, 196 mOsm/kg for ragweed and 424 mOsm/kg for oak pollen. Sequential separation of allergens (Lol p I, Amb a I, Amb a V), enzymes and proteins was demonstrated by specific assays. These observations suggest that the complex stimulus produced immediately after pollen grain hydration at the respiratory mucosal fluid interface is much more intense than previously envisioned. Sequential separation of pollen components has important implications for the production of improved allergenic extracts.

Allergens↗

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

Allergens↗