Ana Vega-Maray

Effects of air pollution on Cup a 3 allergen in Cupressus arizonica pollen grains

BACKGROUND Cupressaceae is a family of plants resistant to airborne contamination, and its pollen is the main cause of winter allergic respiratory diseases, especially in North America, Japan, and Mediterranean countries. Recently, a major allergen from Cupressus arizonica pollen grains, Cup a 3, was cloned and expressed. OBJECTIVE To study the effects of air pollution on the expression of Cup a 3, a thaumatinlike protein, in C. arizonica pollen grains using a combination of transmission electron microscopy and immunocytochemical techniques. METHODS Observations were made in mature and hydrated C. arizonica pollen grains from various regions in Spain with different degrees of air pollution. Specimens were fixed using freezing protocols, and ultrathin sections were incubated with anti-rCup a 3 rabbit polyclonal antibodies. RESULTS Labeling of Cup a 3 was detected in mature and hydrated C. arizonica pollen grains. It was more intense in pollen from polluted air regions, and abundant gold particles were observed as they were released through the pollen grain walls. Furthermore, gold particles remained abundant in the pollen cytoplasm. The labeling was noticeably lower in pollen grains from unpolluted air regions. CONCLUSIONS Cup a 3 is present in the cytoplasm and walls of cypress pollen grains during the air dispersion and hydration stages. The abundance of Cup a 3 in pollen grains under polluted air conditions indicates that these cypresses intensify their activity as a defense from environmental pollution, thus strengthening their allergenicity.

Lipid transfer proteins in Parietaria judaica L. pollen grains: immunocytochemical localization and function

Parietariajudaica L. (Urticaceae) pollen is considered one of the most common causes of allergic respiratory symptoms in the Mediterranean area. The localization of lipid transfer proteins (LTPs) in P. judaica mature and hydrated-activated pollen grains was investigated applying a combination of transmission electron microscopy (TEM) with immunocytochemical methods. Our results show that the content of LTPs in P. judaica pollen grains changes during the process of hydration. The localization of judaica LTPs in the cytoplasm and in the lipid bodies associated with vacuoles demonstrated that LTPs represent primarily intracellular proteins. On the other hand, exposure of the pollen grains to germination medium induced the release of LTPs from the pollen grain. Thus, LTPs are cytoplasmic proteins that are secreted to become available for pollen-stigma interactions and probably induce the IgE antibody responses in allergic patients.

Molecular aerobiology - Plantago allergen Pla l 1 in the atmosphere

INTRODUCTION Exposure to airborne pollen from certain plants can cause allergic disease, but allergens can also be found in non-pollen-bearing fractions of ambient air. This may explain why the allergic response in susceptible patients does not always coincide with the presence and magnitude of airborne pollen counts. Plantago pollen is an important cause of pollinosis in northern Mediterranean countries, but it is difficult to determine its incidence in allergies because Plantago pollen appears in the atmosphere at the same time as grass pollen. OBJECTIVE The study aimed to investigate the relationship between the atmospheric concentration of Pla l 1 aeroallergen and Plantago pollen, and its incidence in a population group. MATERIALS AND METHOD Pollen was sampled using a Hirst-type volumetric trap (Burkard) and Burkard Cyclone sampler (Burkard) for Pla l 1 allergen. Allergen was determined with a Pla l 1-specific ELISA. Serum-specific IgE levels to several plant allergens were measured with the EAST system. RESULTS The aerobiological dynamics of Plantago pollen grains and Pla l 1 did not follow the same trend, whereas the sum of Plantago with some other pollen types showed a more similar behaviour. Of the 118 subjects tested, sera from 52 contained IgE to Plantago pollen, but only 5 were monosensitized. CONCLUSIONS The presence of Pla l 1 in the atmosphere depends not only on Plantago pollen but also on the pollen of other species from the Oleaceae family. Knowledge of the behaviour of allergen Pla l 1 in the atmosphere can help understand better asthma exacerbations associated with aeroallergens.

Detection of airborne Par j 1 and Par j 2 allergens in relation to Urticaceae pollen counts in different bioclimatic areas

BACKGROUND In aerobiological studies, the Parietaria pollen type usually includes all Parietaria and Urtica species found in the area. Given that Urtica is a nonallergenic plant, the pollen counts report incomplete information on the presence of allergens in the atmosphere. Discordance between the pollen concentrations of Urticaceae and allergic symptoms has been observed in patients with pollinosis. OBJECTIVE To compare the Urticaceae pollen counts with the Par j 1 and Par j 2 aeroallergen concentrations from 2 different Spanish geographic areas to determine the allergenic load in the atmosphere. METHODS Hirst-type volumetric traps and Burkard Cyclone samplers were used for pollen counts and aeroallergen capture, respectively. The quantification of Par j 1 and Par j 2 allergens was performed using specific 2-site antibody enzyme-linked immunosorbent assay. Transmission electron microscopy and immunocytochemical techniques were applied to localize these allergens in the orbicules. RESULTS Differences between areas and years were obtained in both pollen and aeroallergen concentrations. Despite the lower pollen counts recorded in Cartagena, higher aeroallergen concentrations were registered compared with Ourense. A lower correlation was achieved between Urticaceae pollen concentrations and aeroallergen levels, with a maximum positive significant correlation (adjusted R2 = 0.466, P < .001). Intense labeling of Par j 1 and Par j 2 proteins was observed in the orbicules, the tapetal membrane, and the tapetal tissue remnants. CONCLUSION This method may be valuable for epidemiologic research to establish correlations between concentrations of Parietaria aeroallergens and clinical symptoms. Therefore, the measurement of aeroallergens should be incorporated into the aerobiological studies with clinical applications.

An approach to the knowledge of pollen and allergen diversity through lipid transfer protein localisation in taxonomically distant pollen grains

The aim of the present study is to localise non‐specific lipid transfer proteins (nsLTPs), obtained from Rosaceae (Prunus persica (L.) Batsch) in pollen grains of taxonomically distant plants, such as Iridaceae (Aristea latifolia G.J. Lewis), Platanaceae (Platanus acerifolia (Aiton) Willd.) and Urticaceae (Parietaria judaica L.), in order to compare pollen and nsLTP diversity. A combination of transmission electron microscopy, immunocytochemical techniques, and rabbit specific antiserum against peach nsLTPs (Pru p 3) were used. Abundant labelling to Pru p 3‐like proteins was observed in the cytoplasm, walls and pollenkitt of A. latifolia pollen grains. The presence of nsLTPs associated with the pollenkitt proves that it takes part in the defence mechanism of pollen grains. The labelling was less intense in the cytoplasm and walls of P. acerifolia. Immuno‐stained gold particles were associated with the vacuoles, lipid inclusions, endoplasmic reticulum and Golgi complex. No significant labelling was found in the P. judaica pollen grains incubated with anti‐Pru p 3 polyclonal antibodies. These results indicate important variations in the nsLTPs of different pollen species. Consequently, no taxonomic relationship between pollen grains and nsLTPs could be established.

Allergenic proteins in Urtica dioica, a member of the Urticaceae allergenic family

BACKGROUND Allergy to the pollen of flowering plant species significantly affects the health of people in many parts of the world. Pollens of related genera usually share common antigens and are often, but not always, cross-reactive. Several studies have shown that Parietaria pollen is one of the most common causes of pollinosis in the Mediterranean area, whereas Urtica has no allergenic significance. OBJECTIVES To report on the localization of Parietaria judaica major allergen in Urtica dioica pollen grains and on the detection of allergenic proteins in U. dioica pollen grains during the hydration-activation process. METHODS A combination of transmission electron microscopy and immunocytochemical methods was used to locate allergenic proteins in U. dioica pollen grains after different periods of hydration-activation using the anti-Par j 1 (4.1.3.) monoclonal antibody and serum samples from allergic patients. RESULTS No significant labeling was noted for Parj 1 allergen after 10, 15, and 20 minutes in the walls and cytoplasm. Slight labeling was observed for allergic proteins in the walls of U. dioica after 10 minutes of hydration, and no significant labeling was found after 15 and 20 minutes of hydration. CONCLUSIONS Immunocytochemical methods confirmed the absence of cross-reactivity between 2 related genera, Parietaria and Urtica, and the lowest allergenic potential of U. dioica.

The Relationship between Tapetum Cells and Microspores Based on Protein Localization in Fraxinus angustifolia (Oleaceae) Pollen Grains

This study contributes to the knowledge of the traffic of substances between tapetum cells and microspores through the localization of pollen proteins during microsporogenesis, by using immunocytochemical techniques with TEM. Because the Oleaceae genera, including Olea and Fraxinus, share a very similar protein profile, an Ole e 1–like protein (which cross‐reacts with the Olea europaea major allergen Ole e 1) was detected in Fraxinus angustifolia pollen grains. From the tetrad to mature–pollen grain stages, distinct labeling intensities were localized in the tapetal cells, anther locule, and orbicules as well as in the microspore and pollen cytoplasms and walls. The localization of this protein in the glycocalyx proves the special role of this layer in ectexine development. At exine deposition stages, the detection of immunogold particles in the anther locule and orbicules can be interpreted as showing an active transfer of these proteins from the tapetum cells to microspores. Moreover, at the bicellular stage, the diffusion of proteins from the pollen cytoplasm to the intine channels indicates their contribution to intine formation. These findings provide new evidence of the role of sporophytic and gametophytic tissues in pollen grain development. Moreover, the Ole e 1–like protein may have an important role in modifying pollen grain walls.

Biometeorological characterization of the winter in north‐west Spain based on Alnus pollen flowering

The timing of pollen appearance in the atmosphere provides a general idea of the flowering onset of plants over a wide area. Woody plants in temperate regions have evolved mechanisms to preserve cells from the risk of frost during adverse weather conditions in the period prior to flowering regulated mainly by temperature. A number of indices have been developed to quantify the rest and heat requirements of temperature, which will enable the plant to adapt to environmental conditions. However, flowering is a dynamic and complex phenomenon and it is difficult to separate individual effects of different meteorological parameters. The use of modified bioclimatic indices could be a major step forward. In this study the Alnus glutinosa flowering in four different areas in north‐western Spain in the period 1995–2003 is examined, and trends identified by means of information gathered by Hirst pollen traps. Temperature plays an important role in the maturation of reproductive organs and pollen production. Comparison with bioclimatic indices showed that temperature during the 25–55 days preceding pollen release was the main controlling factor, and that relationship between flowering time and bioclimatic indicator values differs according to local conditions. In colder areas, rest and heat temperature requirements are greater because the trees need protection over a longer period; in the Mediterranean region of north‐western Spain, the rest temperature requirement and the threshold temperature are both higher than in Eurosiberian areas. Ombrothermic, Continentality and Thermicity indices are thus useful tools for characterizing the various bioclimatic areas of north‐western Spain.

Ultrastructural modifications in the apertural intine of Parietaria judaica L. (Urticaceae) pollen during the early stages of hydration

Hydration and activation of the Parietaria judaica L. (Urticaceae) pollen result in ultrastructural modifications in the apertural region. The particularly distinct intine under the apertures forms convex onci that undergo short but significant and reversible changes during the 10 first minutes of pollen grain hydration and activation. Such changes seem to involve the rough endoplasmic reticulum and show that the oncus is a dynamic structure. This confirms the elasticity of the intine and shows a new pattern of activity in this structure during hydration and activation of pollen germination in vitro.