High IgE sensitization to maize and rice pollen in the highlands of Madagascar

Introduction Maize and rice are two crops constituting the main food supply in many under-developed and developing countries. Despite the large area devoted to the culture, the sensitization to the pollen from these plants is reported to be low and often considered as an occupational allergy. Methods Sixty five Malagasy pollen allergic patients were clinically and immunochemically investigated with regard to maize and rice pollen allergens. Pollen extracts were electrophoretically separated in 1 and 2 dimensions and IgE and IgG reactivities detected upon immunoblotting. Results When exploring the sensitization profile of Malagasy allergic patients to maize and rice pollen, it appears that a high proportion of these patients consulting during grass pollinating season were sensitized to both pollen as revealed by skin prick testing (62 vs. 59%) and IgE immunoblotting (85 vs. 40%). Several clinically relevant allergens were recognized by patients’ serum IgE in maize and rice pollen extracts. Conclusion The high levels of maize and rice pollen sensitization should be related, in this tropical region, to a specific environmental exposure including i) a proximity of the population to the allergenic sources and ii) a putative exacerbating effect of a highly polluted urban atmosphere on pollen allergenicity. Cross-reactivities between wild and cultivated grasses and also between rice and maize pollen are involved as well as some specific maize sensitizations. The presence of dense urban and peri-urban agriculture, in various African regions and worldwide, could be a high environmental risk factor for people sensitive to maize pollen.


Introduction
The highlands constitute, in Madagascar, the main populated area with the presence of the capital town -Antananarivo (around 1.85M inhabitants) -and of Antsirabe (currently around 200, 000 inhabitants). Beside traditional rural agricultural systems, the food supply for such large cities includes, now for decades, peri-urban and urban agricultural zones, as already described in other towns from developing countries [1][2][3]. Rice  Despite its increasing worldwide trends, allergy remains poorly studied in Madagascar [6,7]. In the same West Indian Ocean and South African zones, some studies have reported that the urban population is now often affected and regularly treated [8][9][10].
Among respiratory allergic diseases, allergy to grass pollen represents an important part because grasses are widely distributed. Aerobiological studies in Antananarivo showed that grass pollen is determinant in the urban atmosphere [11].
Sensitization to rice and maize pollen has been clinically investigated [12,13]. They are often considered as an occupational allergy in farmers and field workers [14][15][16] or the result of the documented cross-reactivities with wild grasses [17][18][19]. Reported allergens for pollen of these 2 crops are listed in Table 1. Ten allergens have been described in maize and 7 of them, belonging to the same protein families, were also found in rice pollen. The major allergens β-expansins (groups 1-3 of grass pollen allergens) [20][21][22], profilin (group 12) [23] and polygalacturonase (group 13) [24] were characterized both in maize and rice pollen.
In order to explore the extent of respiratory diseases related to allergy to grass pollen in Madagascar, an epidemiological study was carried out on patients consulting for respiratory disorders in two different centers of Antananarivo [11]. On the basis of a standardized questionnaire, clinical symptoms, skin prick test and correlations of symptoms with pollen airborne content and/or documented pollinating season, 65 allergic patients were selected to quantitatively and qualitatively evaluate some clinical and immunochemical parameters associated to grass pollinosis. In a previous report, we published the results related to 6 selected wild grass pollen and showed that, besides cross-reactivities, sensitization is mainly directed against the local tropical dominant wild grass, Rhynchelytrum repens [7]. To give a complete picture of the prevalence of allergy to grass pollen in Madagascar our results are now extended to cultivated grasses, maize and rice, that show overlapping pollinating periods with wild grasses. The level of sensitization to the pollen of these two main crops was clinically and immunochemically evaluated and was found to be high. The proximity of cultivation areas, close to inhabitants, and/or a highly polluted atmosphere, could be the crucial environmental risk factors in the development of such allergic symptoms in this region.

Methods
This study was conducted in accordance with the principles of the declaration of Helsinki, with institutional approval (from both University of Antananarivo, Madagascar and Hôpital Civil, Strasbourg, France). Written informed consent has been obtained from all patients.

Patient recruitment and patient sera
A previous comprehensive epidemiological study was carried out in the Lung and Allergy Department of the "Institut d'Hygiène Social" and the "Infirmerie de la Gendarmerie du Toby Ratsimandrava" in Antananarivo (Madagascar) on about 1500 individuals suffering from respiratory disorders (consultations and data files analysis).
The follow up during 2 years included the record of clinical symptoms such as respiratory infections, asthma, rhinitis, conjunctivitis, rhino-conjunctivitis, dyspnea but also dermatological disorders such as dermatitis, eczema and urticaria [11]. From this initial cohort, a sub cohort of 65 patients was selected on the basis of (i) the correlation of clinical symptoms with the grass pollinating Page number not for citation purposes 3 season. (ii) skin prick test (reported in [7] and in this paper).and (iii) a standardized questionnaire prepared by physicians with extensive experience in allergic diseases. All grass pollen allergic patients (PAP), 28 males and 37 females, mean age 33 year-old (range: 3 to 65), participated voluntarily in the study. The main symptoms for these patients were rhinitis, conjunctivitis and/or asthma during the flowering season. Some PAP showed food allergy symptoms such as diarrhea, nausea, vomiting associated to rashes. The sera used in this survey are the same than for our previous study [7]. Two additional sera from grass and birch sensitized Caucasian/European patients born in Strasbourg area, (France) were also used [7,25].
These patients had never been in Madagascar. A serum from a nonallergic non-atopic individual was used as control.

Maize and rice pollen material
The pollen was collected directly from the flowers of the 2 crops between March and April. The Zea mays pollen used for most of the experiments of our study was from a Meva cultivar. This variety grows all around the city of Antananarivo and is one of the most cultivated in the island. It is a polyhybride registered as N° 374. This synthetic construction combining 5 South African and a local yellow lines variety has been selected for its resistance to leaves disease and for its adaptation to acidic soils. Its culture is agronomically adapted to an altitude ranging from 800 to 1500 m and to the climate of highlands. Five other varieties of maize pollen were used for 1D-isoelectrofocalisation (IEF) and immunoblot experiments.
Four are from Madagascar: "Paysanne", a local farmer variety, "Volasoa" -also called "Los banös 8227"as well as 2 varieties, created by private companies, "NTS" and "Tombotsoa". The last one is from Europe (Allergon AB, Sweden, W 2552). The rice pollen was a blend from several varieties among the three main seasonal types cultivated in the highlands locally called -from the 1 st to 3 rd seasonal one -respectively: vary aloha, vary vakiambaty and vary siha, including indica and japonica subspecies.

Pollen extracts for skin prick tests (SPTs) and SPTs
Pollen extracts were prepared in the same way than in Ramavovololona et al [7]. Briefly: 1 mL of 0.9% NaCl, 0.3% NaH(CO3)2 was added to 100 mg of each pollen and obtained as previously described [25,26]. SPTs were performed using these pollen extracts and tested on the 65 PAP for the 2 taxa. For the 2 Caucasian/European patients, the grass pollen extract was provided by Stallergènes Laboratories, (Antony, France). A negative control test with saline solution and a positive control test with histamine (1 mg/mL) were included in each set of SPT. SPT response was positively scored if the largest wheal diameter was greater than that was produced by the negative control and at least 70% greater than that was produced by the positive control.

Pollen extracts for IEF and immunodetections
Water soluble extracts were obtained by incubating 30 mg of fresh pollen for 1h at 22°C in 150μL sterile water followed by a centrifugation at 4°C for 15 min at 10,000 g. The supernatants were kept at -20°C. containing 5% v/v Ampholine pH 3.5-9.5 as described above. After the IEF separation, 4 mm-wide strips of the focused gel were cut, incubated in the equilibration buffer (120mM Tris pH 6.8, 12 % SDS) and submitted to SDS-PAGE separation on an 8-18% gradient gel (GE Healthcare). Standard proteins with a range of relative molecular mass (Mr) from 14.4 to 94 kDa (GE Healthcare) was used as Mr markers. One of the 2D gels was silver-stained according to Blum et al. [28] in order to visualize all proteins and the other ones were electroblotted onto a bromide cyanogen-activated nitrocellulose membrane (NCa) [29] using a semi-dry Novablot apparatus (Amersham Biosciences) according to the manufacturer's instructions for 1h at 1 mA/cm 2 . The NCas were then dried, saturated with phosphate buffer saline -0.3% tween 20 and treated as described above. Then, membranes were incubated with APantibodies against either human IgG or IgE.

Statistical analysis
The statistical significance of differences in data was evaluated using Fisher's exact test.

Results
Clinical data of studied PAP are shown in Table 2, Table 3.
Patients were mainly suffering from rhinoconjuntivitis (72%), 53% from asthma and 15% urticaria. Thirty one patients out of 65 (47.7 %) exhibited food allergy symptoms. Percentages of patients tested SPT-positive for maize or rice pollen extracts were both around 60% (Table 4) as compared to 71% for wild grasses. By SPT (Figure 1, A), 44% of patients were found co-sensitized to maize and rice pollen extract and among them, the majority (59%) had a weak positivity. The correlation between SPT to maize and rice pollen was statistically significant (p=0.015).
The specificity of seric IgE (  Table 4). Interestingly, in some patient sera, about 12%, the Ab response to rice pollen protein seemed restricted to IgE isotype since no specific IgG isotype could be detected (data not shown).
Moreover, 19 patient sera (33%) showed IgE reactivity against proteins from both pollen extracts (Figure 1, B Table 1. A 3 rd neutral zone (pI 6.6-7.1) showed also a strong IgE signal for several patient sera (Figure 2, A) and might correspond to isoforms of Zea m 13, the maize pollen polygalacturonase. IgG reactivities were also observed against several maize proteins in a wide pI range (Figure 2, D). By comparison, IEF immunoblots of the rice pollen extract exhibited less heterogeneity of IgE binding (Figure 2, B), but IgG reactivity against this extract was very heterogeneous (Figure 2, E). For the IgE reactivity, the pI range was more restricted and most of the sera gave positive signals in the neutral-basic zone, from pI 7 to 9.5 (Figure 2, B,C), consistent with already described allergens Ory s 1, 2, 3, 7 and 23 ( Table 1). A few patient sera showed IgE binding to proteins with pI between 5.5 and 6.5 as well as in pI range from 6.0 to 6.8 (serum #24), that could correspond to isoforms of Ory s 1.
Page number not for citation purposes 5 IgE reactivities against basic allergens (pI > 9.0), were found only in the maize pollen extract and could correspond to Zea m 3. Sera #66 and #67, from European patients, (called A and B in our previous work [7]) showed a strong IgE binding to basic allergens in maize extract (Figure 2, A) whereas no IgE reactivity was found in rice extract (Figure 2, B).

Discussion
In a general approach to evaluate grass pollen allergy in Madagascar, we clinically and immunochemically explored in a previous study, the IgE reactivity patterns of 65 PAP suffering from symptoms during grass pollination season against six different wild grass pollen [7]. Since wild and cultivated grasses belong to the same Poaceae family, we worked herein on pollen from the 2 most important cereals cultivated in Madagascar: maize and rice. The allergenic sensitization to maize or rice pollen has been reported [12,30] but was shown to be lower as compared to wild grass pollen partly due, at least for maize pollen, to a larger size and heavier weight than wild grass pollen grains resulting in a poor airborne dispersal [31]. In some agricultural regions of Europe [13], the prevalence of sensitization (evaluated by SPT) of PAP attending departments of allergy in hospitals between March and June is about 16% in region where maize is cultivated in medium to high density and was up to 88% for wild grasses. We showed here, by SPT, that the sensitization to maize or rice pollen in the highlands of Madagascar is very high, reaching 60% of patients selected upon clinical symptoms during grass pollinating season. The prevalence of sensitization to 6 different wild grasses tested individually by SPT were reported to be of the same order of magnitude, between 45 to 55% when considered taxon per taxon [7] but 71% for any wild grass pollen. Thus, the epidemiological data are somewhat different with what was found in Europe. Although SPT data might be in favor of cross-reactivity between maize and rice pollen, this trend was not confirmed by immunoblot results that showed more reactivities to maize pollen (85%) than to rice pollen (40%). Since allergen families are shared between maize and rice as well as between wild and cultivated grass pollen, cross-reactivities were expected [16,17,24,[32][33][34]. However, some specific sensitization can also occur in maize pollen allergy and rather associated to Page number not for citation purposes 6 occupational allergy [14,15]. An absence of cross-reactivity is thus in favor of a genuine specific sensitization as a consequence of a sustained and specific exposure. The IgE bindings observed by immunoblot reflect clinically relevant and irrelevant IgE reactivities whereas SPT evaluates IgE reactivities able to induce mediator release, and thus being closer to clinical relevance. Whether maize (pollen and/or corn kernel) could induce more clinically irrelevant IgE-binding protein as compared to rice (pollen and/or grain) has not yet been studied. Interestingly the high immunoblot reactivity against maize pollen was observed within IgE as well as IgG isotypes whereas in 12% of patients, anti-rice pollen Ab response seemed to be restricted to IgE isotype. This may reflect differential sensitization processes.
Cross-reactivities also exist between pollen and grain food that may lead to potential food allergy symptoms. Indeed, 47.7% of the PAP studied herein also display food-allergy symptoms ( Table 2) and 43% express IgE against proteins from ground rice extract (unpublished data). Fonseca et al [35] showed different IgE binding proteins profiles in the maize seed extracts depending of the cultivars. In our study, on pollen extracts from 5 selected maize cultivars, only small differences in IgE binding protein profiles were visualized. Consequently, it appeared worthwhile to use the largely cultivated local Meva cultivar for 2D separations. Regarding maize pollen proteins and allergens, very few 2D separation studies were available till now [15,32]. The IgE binding pattern of the studied serum showed a larger coverage of the maize pollen allergens than what was reported with sera from PAP exposed to maize pollen in greenhouses [32]. Four IgE binding proteins detected could correspond to already characterized allergens: i) the very abundant Zea m 1, known as major pollen allergen of maize [22] and reaching 4% of the extractable protein content [36], ii) the polygalacturonase Zea m 13, iii) Zea m 4, a reticulin oxidase and iv) Zea m 22, an enolase also present in maize seed [35]. Allergens belonging to these protein families have also been described in some other wild and cultivated grass pollen [24] (www.allergome.org). Beside these 4 known proteins, described as the most prominent maize pollen allergens [16,24], our 2D immunoblots revealed 3 other IgE binding protein zones that remain to identify. No indication of group 5 allergens was evidenced in agreement with some report claiming its absence in maize [34]. Controversially, on its own, the study by van Ree et al [37] showed that group 5 allergen is present in Zea mays pollen. Furthermore, links between Lol p5, Ant o 5, Phl p 5, Sec c 5, Phr a 5, Fes r 5 and Dac g 5 were noted by Mohapatra et al [38], with dominating Pooideae species, compared to Arundineae (Phr a 5) and Panicoideae (Zea m 5). Group 2 allergens were also questioned in maize pollen [34] and, however, reported upon DNA sequencing [39].
Our study suggests that cereal pollen allergy -mainly for maize one -should be taken into account more carefully. Indeed, SPT and IgE immunoblots results revealed a high prevalence in Madagascar similar to what is observed for wild grass pollen. Several hypotheses may be raised to understand the results. Firstly an increase of exposure to maize pollen may be incriminated. In Madagascar, like in many other African countries, the part of the maize as cereal feeding source permanently increased. Between 1970 and 2000, the increase of cultivated surfaces was more than 100% for the maize vs. 45% for the local Sorghum [40]. Moreover, peri-urban and urban agricultures are determinant in food supply for many huge cities [41] and in Antananarivo, people are living very close to corn fields used for supplemental food supply. Population is, consequently, more exposed to maize pollen source. In such a way the risk of allergenic sensitization is higher even for non atopic individuals. Indeed, each maize plant produces copious pollen and even though the size of the grains (around 100 µm) could limit its dispersal, it can reach more than 1000 m downwind from the maize crops sources. In most of the cases maize pollen deposition is

Competing interests
Authors declare no competing interests.

Acknowledgments
We would like to warmly thank S. Rakotomamonjy, in charge of a national maize breeding program in Madagascar, for giving precise information on the different maize varieties. We also thank S.
Descorps-Declère, Pasteur Institute, Paris, France, for his precious help in statistical calculation.     and Mr (kDa, on the right side) are indicated for each gel    SPT response was scored as + positive, if the largest wheal diameter was comprised between 70 to 80% greater than that was produced by the positive control, ++ between 80 to 90% and +++ between 90 to 100%Results of IgE immunoblot (see reactivity figure 2) are semi quantitatively expressed according to the intensity of the reaction as: -:

Tables and figures
negative; + positive low; ++ medium; +++ very high.    : 2D analysis of the water-soluble proteins from Zea mays pollen. Pollen extract from Zea mays was submitted to an initial IEF separation followed by SDS-PAGE separations. The gels was either silver stained (A) or transferred on NCas and incubated with from pollen-sensitized patient serum #60. IgG (B) or IgE (C) binding were revealed using heavy chain specific Ab coupled to alkaline phosphatase followed by substrate. Boxes on each figure indicate groups of allergens recognized by IgE PAP. pI values (at the top) and Mr (kDa, on the right side) are indicated for each gel