Review Article
Austin J Allergy. 2015; 2(1): 1019.
Amaranthus Pollen Allergens: Protein Diversity and Impact on Allergy Diagnosis
Hasnain SM1*, Alsini HA1, Gad El-Rab MO2, AlFrayh AR2 and Alaiya AA3
1Department of Cell Biology, King Faisal Specialist Hospital and Research Centre, Saudi Arabia
2College of Medicine, King Saud University, Saudi Arabia
3Stem Cell & Tissue Re-Engineering Program, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
*Corresponding author: Hasnain SM, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
Received: November 24, 2015; Accepted: December 17, 2015; Published: December 21, 2015
Abstract
Allergenic weeds dominate the pollen air flora (> 80%) of Saudi Arabian environment. Two species viz Amaranthus viridis (Av) (Slender or Green Amaranth) and A. lividus (Al) are the most prevalent components of this flora. Although all Amaranthus species appear to be allergenic and a potential cause of respiratory allergy, neither a diagnostic extract nor raw pollen grains for Av is available commercially for comparative study on patients.
Thus, in order to determine IgE mediated sensitization of Av and to observe cross-reactivity patterns with other species, an allergological study was conducted using seven Amaranthus species. Row pollen of 5 Amaranthus species were acquired from two commercial sources (Greer USA, Allergon Europe) while Av & Al pollen was collected indigenously. Allergenic extracts were prepared using pollen grains from both sources in buffered saline. Skin Prick Test (SPT) was conducted on 132 allergic patients using all seven extracts.
Protein separation of all seven Amaranthus species was conducted by SDSPAGE. The results indicate that the species of Amaranthus vary in their protein profiles with a pattern of cross SPT reactivity between the species. However, as the exposure takes place with prevalent pollen form Av and Al, the commercial extracts using species not present in the region may not be fully relevant to the patients for diagnosis and immunotherapy
Keywords: Allergy; Pollen; Amaranthus viridis; Diagnosis; Amaranthus species; Protein diversity
Abbreviations
SDS-PAGE: Sodium Dodecyl Sulphate Polyacrylamide Electrophoresis; AV: Amaranthus viridis; SPT: Skin Prick Test; RAC: Research Advice Council
Introduction
Allergy and Asthma in both children and adult can be caused by many allergenic pollen grains from weeds, trees and grasses [1]. World allergenic pollen flora varies in their nature and quantity from place to place and fluctuates with geography and climate.Bronchial Asthma is very common allergic disease occurring in all age groups, particularly children, all over the world and the trend of asthma prevalence in both developed and developing countries are increasing over the last 30 years [2].
Environmental factors are known to play an important role in the elicitation of asthma in genetically predisposed individuals. Although there has also been an increase in the awareness among doctors to diagnose asthma, a combination of various other factors may also be involved in the increased prevalence of asthma [3]. The soil and climate of KSA was once considered unfavorable for plant growth. A large number of plants have been introduced to the kingdom in recent years [4].
The genus Amaranthus consist of several species. It is an allergenic weed shedding pollen in the air throughout the year in Saudi Arabia with peaks in autumn months. There are a number of Amaranthus species in Saudi Arabia as listed in (Table 1). Each of them, with some synonym, is known by a common name as well. Both, the common and synonymic names are also presented in this table. However, the dominant species on the ground and frequently encountered pollen in the air belongs to A. viridis [5] (Figure 1).
Scientific name
Synonym
Common name
Amaranthus albus
A. var. pubescens, A. graecizans auct. non, A. var. pubescens Uline & Bray [7]
White Pigweed, Prostate Pigweed, Pigweed Amaranth, White Amaranth.
A. caudatus
A. edulis Speg. Amaranthus leucocarpus (S.Watson.),[8]
Pendant Amaranth, Love-lies-Bleeding, Tassel Flower, Quilete.
A. graecizans ssp sylvestris
A.graecizans [9,10]
A.blitum
A. graecizans
A. angustifolius A.albus [9,10]
Prostate Pigweed
A. hybridus ssp hybridus
A. chlorostachys [10].
Smooth Amaranth, Smooth Pigweed, Red Amaranth, Slim Amaranth.
A. hybridus ssp crutentus
A. crutentus [10].
Purple A., aka, Red A, Mexican grain A, Caterpillar.
Amaranthus palmeri
[11]
Palmer's amaranth, palmer pigweed, careless weed
A. lividus **
A. blitum, A. ascendens [10].
Purple Amaranth
A. spinosus
[9,10]
Spiny Amaranth, Prickly Amaranth, Thorny Amaranth, Spiny Amaranthus.
Amaranthus tricolor
A. tristis, A. mangostanus [10]
Joseph's-coat
A. viridis *
A. gracilis Desf. [8]
Slender Amaranth, Green Amaranth
*Most common in Saudi Arabia (+++) (as per growth pattern)
**Less common in Saudi Arabia (++) (as per growth pattern)
All others species are rare and sporadic.
Table 1: Amaranthus Species in Saudi Arabia [7-11].
Figure 1: Amaranthus viridis weed.
Survey of the Amaranthus species was made in the deserted countryside of Saudi Arabia in view to observe the growth pattern and collection. It was revealed that there are some species, which are very close to Amaranthus but appeared not to be Amarathus viridis.
The allergenic extract, prepared using Amaranthus pollen, are used for the diagnosis of respiratory allergic disorder to this pollen and like-wise, the treatment vaccine for immunotherapy is prepared using pollen extract.
It appears that A. viridis extract is not included in the diagnostic profile in Saudi Arabia by the clinicians and instead, unrelated imported/commercial extract of other Amaranthus spp. under the common name of Pigweeds are included. This is likely to result in false negative reactivity in those patients who are exposed to A. viridis. There are only up to 30% cross-reactivity within the weeds pollen allergy but no such cross-reactivity has been documented within all Amaranthus pollen allergens [6]. Apart from the cross reactivity, treatment by immunotherapy may not be successful unless precise molecular relation between offending allergen and desensitized allergens are established.
Materials and Methods
Collection of indigenous Amaranthus
Two Amaranthus species (Av, Al) were primarily collected from Riyadh, Jeddah, Taif and Najran regions. Majority of these species were found growing in parklands, home backyard, home gardens, lawns etc
Several lots of flowering Amaranthus were collected at different time intervals from different places. All collections were properly dried. After drying the collected plants, anthers were separated. The separated anthers were further dried, treated and teased with acetone and 95% alcohol, centrifuged and dried as raw material, stored at 4oC and used in the preparation of extracts. Pollen samples showing more than 90% purity were included in the investigations.
Pollen from commercial sources
Based on the international availability, commercial pollen grains of the following species were purchased from various commercial suppliers in Europe and USA:
These included: Amaranthus palmeri, Amaranthus tuberculatus, Amaranthus retroflexus, Amaranthu shybridus (Greer Laboratory, USA), (Amaranthus retroflexus, Amaranthus tamariscinus. Allergon Company, Europe) [7-11].
Pollen protein extraction
Both collected and commercial pollens were defatted with excess of diethyl ether / n-butanol to achieve maximum removal of lipids and pigments. Antigenic protein was extracted from the defatted pollen with 1:10 weight per volume (w/v) concentration. The extract was prepared in Phosphate Buffered Saline [12] (10 mM PBS pH 8 at 40oC for 72 hrs). After the extraction, it was centrifuged at 4000 rpm for 15 min and the supernatant was dialyzed (mol. wt. cut limit: 3500) exhaustively against 85 % PBS, lyophilized by freeze drying system in small aliquots and stored at –200oC and reconstituted, when and as required. Protein content of each extract was determined by Bradford method [13]. The extracts were sterilized by bacterial filter by passing through 0.45 mm and 0.22 mm filter using Millipore filter units. For in vivo SPT, 50% glycerinated extracts were prepared. The purity and sterility for each extract was tested using Brain Heart Infusion Agar and Blood Agar for at least 15 days at 37oC. The test was negative indicating no contamination.
Sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE)
The procedure outlined by Laemmli [14] was followed. SDSPAGE was carried out using 12 % polyacrylamide gel using Mini Electrophoretic Apparatus (Bio Rad). Extracts with varying protein concentrations were used in loading. The gels were calibrated with marker proteins with molecular weights of 10, 15, 20, 25, 37, 50, 75, 100, 150, 250 kD (Bio-Rad). The gels were stained using staining solution (10% glacial acetic acid, 0.25 % Commassie brilliant Blue in 45% methanol), then destained for varied periods until protein bands appeared clear. After destaining, the gels were scanned.
Skin Prick Test (SPT)
Skin prick tests were performed on 39 allergic patients and 10 healthy control subjects, attending the Allergy clinic at King Khalid University Hospital, Riyadh. Phosphate buffered saline and histamine were also tested as negative and positive control respectively. The skin response was observed after 15-20 minutes of the test and graded as per the criteria:
<3mm Negative,
=3mm Low Positive,
5-10mm Moderate Positive, and
<10mm Strong Positive
Serum samples
Venous blood was drawn from 17 skin test positive patients and sera was separated by centrifugation and stored at -20ºC in small aliquots for further use. Blood samples from 10 healthy volunteers were also collected to act as control. ARAC approved consent form was signed by each patient for SPT and blood draw.
Immunoblot
Electrophoretic transfer of proteins to PVDF membrane following the method of Towbin et al. [15]. Proteins separated by SDS-PAGE were electrophoretically transferred to a 0.45µm Polyvinylidene Difluoride (PVDF) membrane for immune detection of IgE in serum of sensitized subjects bound to allergenic proteins. Highly positive sera from hypersensitive patients were used to determine the IgE binding fractions in pollen extracts.
PVDF membrane (0.45 µm) of the size of the gel was soaked in the transfer buffer, Tris-glycine buffer (25mM Tris, 200 mM Glycine, 20% methanol, pH8.3) an hour before the transfer of proteins. Proteins were then blotted to membrane by electro transfer using the transfer buffer at 30mA at 4°C for overnight.
The un-reacted sights on the membrane were blocked with 5% non fat milk in 0.05 % Tween20 Phosphate Buffered Saline (PBST) at room temperature for 1 hour. Washed by PBST then, membrane is incubated with pooled sera of positive individual. Pooled sera from healthy individual showing negative skin reactivity were used as control.
In all incubations serum was diluted in the ratio of 1:500 using PBS containing 0.05 % Tween20. Membrane was washed thoroughly using 0.05% PBST. After washing the membrane was blocked by non fat milk (5%). Membrane was incubated with antihuman IgE peroxidase conjugate (Sigma) in the ratio of 1:10000 in 0.05% PBST for I hour at room temperature. The membrane then washed thoroughly 4 times by washing buffer 0.05% PBST. After the last wash the membrane was washed by PBS to remove all the Tween. Membrane was developed in dark room after ECL super signal incubation for 5 minutes.
Results
Protein estimation
The Protein concentration for each sample was:
1. Amaranthus viridis – 1.172 µg/µl
2. Amaranthus lividus – 0.637 µg/µl
3. Amaranthus retroflexus (Allergon) – 1.598 µg/µl
4. Amaranthus retroflexus (Greer) – 2.379 µg/µl
5. Amaranthus tuberculatus (Greer) – 0.852 µg/µl
6. Amaranthus hybridus (Greer) – 0.941 µg/µl
7. Amaranthus palmeri(Greer) – 1.300 µg/µl
Sodium Dodecyl Sulphate Polyacrylamide Electrophoresis (SDS-PAGE)
Figure 2 - A 12 % SDS-PAGE, shows equal volumes of samples were loaded, the highest concentration was in L4 (Amaranthus retroflexus ) from Greer company which means that the purity of the pollens were 100%.
Figure 2: 12% SDS-PAGE of different Amaranthus Species.
Remarkable differences in protein profile between the indigenous species (L1, L2) and the commercial ones (L3- L7) are shown in the gel. The indigenous samples showed bands at lower molecular weight ranging between 36 kD& 14 kD, while the commercial species showed proteins at higher ones. The indigenous Amaranthus viridis has bands at 36, 33, 31, 24, 20 and 15 kD, and Amaranthus lividusat 36, 33, 31, 29, 20 and 15 kD. The commercial ones from Greer Amaranthus retroflexus showed bands at 87, 70, 48, 40, 36, 32, 28, 26, 20, 17 and 14 kD. Amaranthus tuberculatus at 87, 70, 40, 36, 24 and 17 kD. Amaranthus hybridus at 87, 70 and 40 kD. Amaranthus palmeri at 87 and 70 kD.
SPT results
Out of 132 consecutive patients attending the Allergy clinic at King Khalid University Hospital, Riyadh (KKUH), sixty five patients (65/47.1%) reacted positively to Amaranthus extracts, both commercial and locally prepared. The skin test reactivity of the 39 patients, who participated in the study are presented in (Table 2 and Figure 3).
*(wheal diameter in mm)
ALLERGENS
Patient No.
1 A. viridi (local)*
2 A.lividus (local)*
3 A. retroflexus (Allergon)*
4 A. retroflexus (Greer)*
5 A. tuberculatus (Greer)*
6 A. hybridus (Greer)*
7 A. palmeri (Greer)*
1
3
4
7
6
6
7
7
2
4
-
-
4
-
-
-
3
8
7
7
5
9
5
8
4
5
5
4
10
6
4
4
5
7
-
8
10
8
7
6
6
4
-
-
7
4
4
4
7
6
7
-
-
-
-
-
8
-
-
4
7
4
4
4
9
-
-
-
4
5
-
6
10
3
3
5
8
10
6
5
11
5
-
6
5
5
6
6
12
7
5
4
8
7
4
7
13
10
10
14
10
10
10
9
14
5
3
8
9
7
6
5
15
4
3
5
7
6
6
5
16
-
-
-
-
4
3
4
17
-
-
3
5
5
5
4
18
5
4
8
15
10
12
8
19
4
4
6
9
6
5
6
20
-
-
-
6
4
4
3
21
5
4
4
4
4
4
4
22
6
6
14
9
9
12
8
23
9
8
5
7
6
5
5
24
4
5
10
6
5
5
7
25
-
-
-
7
5
3
4
26
3
3
3
7
4
6
5
27
4
-
5
3
-
4
6
28
4
4
6
8
6
6
7
29
5
4
7
8
6
5
6
30
3
-
-
5
3
3
3
31
-
4
5
6
8
6
4
32
10
6
4
16
6
3
4
33
7
6
4
8
9
9
9
34
7
5
12
10
14
10
9
35
4
3
5
7
6
6
5
36
4
3
4
7
6
3
4
37
-
-
3
6
5
5
6
38
6
4
-
4
4
5
3
39
7
5
5
4
4
4
4
Table 2: Skin test reactivity of 39 patients to seven Amaranthus extracts.
Figure 3: Mild and Strong SPT reactivities of various Amaranthus extracts.
In the whole group (39), 31(76.92%) reacted to A. viridis (indigenous species), while 26 patients (66.66%) reacted to A. lividus (indigenous species).
Twenty five patients (64.10%) reacted to both local extracts. Four patients showed strong reactions to the local Amaranthus allergens.
Reactions to the other Amaranthus species were as follows: 30(76.92%) to A. retroflexus (Allergon), 37(94.87%) to A. retroflexus (Greer), 36(92.30%) to A. tuberculatus (Greer), 36(92.30%) to A. hybridus (Greer) and 37(94.87%) to A. palmeri (Greer).
Immunoblot
1. Immunobloting of serum samples (17 patients), we found that 94% of the Amaranthus sensitized (IgE mediated positive SPT) individuals have IgE-binding antibodies to Amaranthus viridis (Indigenous) pollen extract. The major Amaranthus allergen defined as binding IgE from most subjects is 52, 31 Kda. Other IgE-binding allergens were found at 38, 20, 17and 14Kda. 3.
2. All patients reacted to proteins at 31 Kda and 52 Kda. It was interesting to note that indigenous extracts contained 31 Kda & 52 Kda proteins and out of 17 patients, 16 (94%) reacted to indigenous extract (A. viridis). However, one patient who did not react to indigenous (A. viridis, possibly an error) reacted to 31 Kda protein of other indigenous (A. lividus). Likewise, two patients, who did not react to A. lividus (patient no. 6&7), reacted to A. viridis. Therefore, it was 100% immune-reactivity towards two species of indigenous Amaranthus species (Figure 4).
Figure 4: Immunoblot showing Amaranthus species specific IgE binding
fractions of antigenic extracts probed with the sera of SPT positive patient
(p13).
3. Surprisingly, all patients showed allergenicity to sample no.4 (Figure 5) and 76.47 % showed allergenicity to sample no. 3, species which are not found in KSA.
Figure 5: Individual sensitivity patterns to Amaranthus retroflexus extract on
immunobloting
Discussion
This study has provided much important information as regards to Amaranthus allergens that are prevalent in the Kingdom of Saudi Arabia and those which are imported into the country for diagnostic and therapeutic reasons.
There are only a handful of companies in the world, mainly in Europe and North America, producing Amaranthus extract for diagnostic and therapeutic use. Most imported extract belong to species which are not found in the Kingdom of Saudi Arabia. The list of commercial Allergenic Pollen Powder (& aqueous extracts) revealed that none of them produce extracts for SPT using A. viridis. Our literature search also indicates that there are no commercial suppliers of A. viridis pollen powder and extract. This is an interesting observation because A. viridis is the dominant species in Saudi Arabia while no suppliers has access to this pollen in the world market to date.
“Amaranthus extract” means extract from any species or variety of Amaranthus which may or may not include the viridis species. Some of the Amaranthus are known as: Amaranthus lividus (Purple amaranth), Amaranthus palmeri (Careless weed), Amaranthus retroflexus (Pigweed, Rough (Redroot)), Amaranthus viridis (Slender amaranth, Green amaranth) etc.
There may be clinics and hospitals in the Kingdom getting commercial “Amaranthus extract”, but the question is that they need to know whether the species they are using is available in Saudi Arabian environment and how prevalent they are? Are patient exposed to the same species where they live??
The result has indicated that there is cross-reactivity between some species of Amaranthus and that is the reason that A. retroflexus, a species not found in Saudi Arabia, reacted in most patients [16]. The A. retroflexu was purchased from Greer company in the United States swas highly purified. The allergen extract prepared in our Lab using the same technique, as used for others, gave a high protein content compared to others. However, when the main allergen in our environment is identified, it is questionable to use cross-reactive allergens [17].
It has been noted (personal communication with many Allergists in the Kingdom) that patients undergoing immunotherapy with “Pollen allergens” are not successfully treated. The probable reason may be the precise molecular relationship to desensitize the patient and the causative allergenic determinants may be different from the determinants in immunotherapy products. In the present study, we found a high degree of reactivity to Amaranthus viridis with their IgE binding allergenic proteins at 31 Kda and 52 Kda (Figure 6). Some of the commercial extract also contained the same allergenic proteins [18].
Figure 6: Immunoblot showing Amaranthus viridis specific IgE binding
fractions of antigenic extracts when probed with the sera of positive patients
[1-9] and control sera [10,11].
Our study also revealed that though there are a good number of individual who are SPT positive but in immunoblot, even a higher degree of reactivity was recorded.
Conclusion
We therefore conclude that despite cross reactivity within Amaranthus species efforts should be made to use Av or Al extracts for diagnosis and treatment especially patients in Saudi Arabia.
Acknowledgment
This Research was funded by King Abdul-Aziz City for Science and Technology (KACST) under a Research Grant ARP 27-11. The informed consent form was approved by the Research Advice Council (RAC) of KFSH&RC in Riyadh. Authors also acknowledge the support of Research center at King Faisal Specialist Hospital in Riyadh and Cheryl Mijares-Oblea for secretarial help.
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