IgE cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens

Background The subtropical Bahia grass (Paspalum notatum) is an important source of pollen allergens with an extended season of pollination and wide distribution in warmer climates. The immunological relationship between pollen allergens of Bahia grass and temperate grasses is unresolved. Methods Serum IgE reactivity of grass pollen-allergic patients with Bahia, Ryegrass and Bermuda grass pollen extracts and their purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA, inhibition ELISA, basophil activation by flow cytometry and molecular modeling. Results Differences in antibody recognition of allergenic components between Bahia grass and Ryegrass pollen were observed by immunoblotting. Eight grass pollen-allergic patients from a temperate region showed greater serum IgE reactivity with Ryegrass pollen than Bahia grass by ELISA. For seven of these sera, Ryegrass pollen inhibited IgE reactivity with Bahia grass pollen but not the converse. For 51 sera from grass pollen-allergic patients in this temperate region, IgE reactivity with Lol p 1 was greater than Pas n 1 or Cyn d 1. IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1, but Pas n 1 IgE reactivity was inhibited by Lol p 1. Two group 1 grass pollen allergen-specific mAb distinguished between temperate and subtropical grass pollens. Basophil activation for three patients tested was greater by Ryegrass pollen than Bahia or Bermuda grass, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, two patients from a subtropical region had higher serum IgE reactivity with Bahia grass pollen than Ryegrass and Bahia grass pollen inhibited IgE reactivity with Ryegrass. A structural model of Pas n 1 showed amino acids implicated in IgE epitopes of other group 1 allergens were juxtaposed on the surface. Conclusion Allergens from subtropical Bahia grass pollen, including Pas n 1, share antigenic determinants with temperate grass pollen allergens, but patients exhibit higher serum IgE reactivity to their locally predominant grass pollen. Basophil activation by Bahia grass pollen and Pas n 1 in patients from a temperate climate indicates clinically relevant cross-sensitization between temperate and subtropical grass pollens.

Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the β-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29 kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.

Bahia grass pollen specific IgE is common in seasonal rhinitis patients but has limited cross-reactivity with Ryegrass

Background: Perennial Ryegrass is a major cause of rhinitis in spring and early summer. Bahia grass, Paspalum notatum, flowers late into summer and could account for allergic rhinitis at this time. We determined the frequency of serum immunoglobulin (Ig)E reactivity with Bahia grass in Ryegrass pollen allergic patients and investigated IgE cross-reactivity between Bahia and Ryegrass. Methods: Serum from 33 Ryegrass pollen allergic patients and 12 nonatopic donors were tested for IgE reactivity with Bahia and Ryegrass pollen extracts (PE) by enzyme-linked immunosorbent assay (ELISA), western blotting and inhibition ELISA. Allergen-specific antibodies from a pool of sera from allergic donors were affinity purified and tested for IgE cross-reactivity. Results: Seventy-eight per cent of the sera had IgE reactivity with Bahia grass, but more weakly than with Ryegrass. Antibodies eluted from the major Ryegrass pollen allergens, Lol p 1 and Lol p 5, showed IgE reactivity with allergens of Ryegrass and Canary but not Bahia or Bermuda grasses. Timothy, Canary and Ryegrass inhibited IgE reactivity with Ryegrass and Bahia grass, whereas Bahia, Johnson and Bermuda grass did not inhibit IgE reactivity with Ryegrass. Conclusions: The majority of Ryegrass allergic patients also showed serum IgE reactivity with Bahia grass PE. However, Bahia grass and Ryegrass had only limited IgE cross-reactivity indicating that Bahia grass should be considered in diagnosis and treatment of patients with hay fever late in' the grass pollen season.

VH gene usage in immunoglobulin E responses of seasonal rhinitis patients allergic to grass pollen is oligoclonal and antigen driven

Background: IgE is the pivotal-specific effector molecule of allergic reactions yet it remains unclear whether the elevated production of IgE in atopic individuals is due to superantigen activation of B cell populations, increased antibody class switching to IgE or oligoclonal allergen-driven IgE responses. Objectives: To increase our understanding of the mechanisms driving IgE responses in allergic disease we examined immunoglobulin variable regions of IgE heavy chain transcripts from three patients with seasonal rhinitis due to grass pollen allergy. Methods: Variable domain of heavy chain-epsilon constant domain 1 cDNAs were amplified from peripheral blood using a two-step semi-nested PCR, cloned and sequenced. Results: The VH gene family usage in subject A was broadly based, but there were two clusters of sequences using genes VH 3-9 and 3-11 with unusually low levels of somatic mutations, 0-3%. Subject B repeatedly used VH 1-69 and subject C repeatedly used VH 1-02, 1-46 and 5a genes. Most clones were highly mutated being only 86-95% homologous to their germline VH gene counterparts and somatic mutations were more abundant at the complementarity determining rather than framework regions. Multiple sequence alignment revealed both repeated use of particular VH genes as well as clonal relatedness among clusters of IgE transcripts. Conclusion: In contrast to previous studies we observed no preferred VH gene common to IgE transcripts of the three subjects allergic to grass pollen. Moreover, most of the VH gene characteristics of the IgE transcripts were consistent with oligoclonal antigen-driven IgE responses.

A novel grass pollen allergen mimotope identified by phage display peptide library inhibits allergen-human IgE antibody interaction

The aim of this study was to investigate the molecular basis of human IgE-allergen interaction by screening a phage-displayed peptide library with an allergen-specific human IgE-mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE-allergen interaction. Biopanning of a phage-displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12-mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents. © 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 μg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

The effects of high temperature on cardiovascular admissions in the most populous tropical city in Vietnam

This study examined the short-term effects of temperature on cardiovascular hospital admissions (CHA) in the largest tropical city in Southern Vietnam. We applied Poisson time-series regression models with Distributed Lag Non-Linear Model (DLNM) to examine the temperature-CHA association while adjusting for seasonal and long-term trends, day of the week, holidays, and humidity. The threshold temperature and added effects of heat waves were also evaluated. The exposure-response curve of temperature-CHA reveals a J-shape relationship with a threshold temperature of 29.6 °C. The delayed effects temperature-CHA lasted for a week (0–5 days). The overall risk of CHA increased 12.9% (RR, 1.129; 95%CI, 0.972–1.311) during heatwave events, which were defined as temperature ≥ the 99th percentile for ≥2 consecutive days. The modification roles of gender and age were inconsistent and non-significant in this study. An additional prevention program that reduces the risk of cardiovascular disease in relation to high temperatures should be developed.

Structural characteristics of a giant tropical liana and its mode of canopy spread in an alien environment

To circumvent the practical difficulties in research on tropical rainforest lianas in their natural habitat due to prevailing weather conditions, dense camouflaging vegetation and problems in transporting equipment for experimental investigations, Entada pursaetha DC (syn. Entada scandens Benth., Leguminosae) was grown inside a research campus in a dry subtropical environment. A solitary genet has attained a gigantic size in 17 years, infesting crowns of semi-evergreen trees growing in an area roughly equivalent to 1.6 ha. It has used aerially formed, cable-like stolons for navigating and spreading its canopy across tree gaps. Some of its parts which had remained unseen in its natural habitat due to dense vegetation are described. The attained size of this liana in a climatically different environment raises the question as to why it is restricted to evergreen rainforests. Some research problems for which this liana will be useful are pointed out.

Species-area and species-individual relationships for tropical trees: A comparison of three 50-ha plots

1 Species-accumulation curves for woody plants were calculated in three tropical forests, based on fully mapped 50-ha plots in wet, old-growth forest in Peninsular Malaysia, in moist, old-growth forest in central Panama, and in dry, previously logged forest in southern India. A total of 610 000 stems were identified to species and mapped to < Im accuracy. Mean species number and stem number were calculated in quadrats as small as 5 m x 5 m to as large as 1000 m x 500 m, for a variety of stem sizes above 10 mm in diameter. Species-area curves were generated by plotting species number as a function of quadrat size; species-individual curves were generated from the same data, but using stem number as the independent variable rather than area. 2 Species-area curves had different forms for stems of different diameters, but species-individual curves were nearly independent of diameter class. With < 10(4) stems, species-individual curves were concave downward on log-log plots, with curves from different forests diverging, but beyond about 104 stems, the log-log curves became nearly linear, with all three sites having a similar slope. This indicates an asymptotic difference in richness between forests: the Malaysian site had 2.7 times as many species as Panama, which in turn was 3.3 times as rich as India. 3 Other details of the species-accumulation relationship were remarkably similar between the three sites. Rectangular quadrats had 5-27% more species than square quadrats of the same area, with longer and narrower quadrats increasingly diverse. Random samples of stems drawn from the entire 50 ha had 10-30% more species than square quadrats with the same number of stems. At both Pasoh and BCI, but not Mudumalai. species richness was slightly higher among intermediate-sized stems (50-100mm in diameter) than in either smaller or larger sizes, These patterns reflect aggregated distributions of individual species, plus weak density-dependent forces that tend to smooth the species abundance distribution and 'loosen' aggregations as stems grow. 4 The results provide support for the view that within each tree community, many species have their abundance and distribution guided more by random drift than deterministic interactions. The drift model predicts that the species-accumulation curve will have a declining slope on a log-log plot, reaching a slope of O.1 in about 50 ha. No other model of community structure can make such a precise prediction. 5 The results demonstrate that diversity studies based on different stem diameters can be compared by sampling identical numbers of stems. Moreover, they indicate that stem counts < 1000 in tropical forests will underestimate the percentage difference in species richness between two diverse sites. Fortunately, standard diversity indices (Fisher's sc, Shannon-Wiener) captured diversity differences in small stem samples more effectively than raw species richness, but both were sample size dependent. Two nonparametric richness estimators (Chao. jackknife) performed poorly, greatly underestimating true species richness.

Structural, physiological and molecular characterisation of the Australian native resurrection grass Tripogon loliiformis (F.Meull) C.E.Hubb during dehydration and rehydration

There is an urgent need to develop crops that can withstand future climates. Results from this thesis demonstrated that a native Australian resurrection grass exhibits structural, physiological and metabolic strategies to tolerate drying. These strategies may be utilized for the generation of stress tolerant crops.

Tropical prawn trawl bycatch of fish and seasnakes reduced by Yarrow Fisheye Bycatch Reduction Device

Reducing unwanted trawl bycatch is actively encouraged in Australia, particularly in prawn trawl fisheries. We tested the performance of a Bycatch Reduction Device, the Yarrow Fisheye, during two periods of commercial fishing operations in Australia's Northern Prawn Fishery, by comparing the catches of paired treatment and control nets. We compared the catch weights of the small fish and invertebrate bycatch, and the commercially important tiger prawns, from 42 trawls in 2002. The Yarrow Fisheye reduced the weight of small bycatch by a mean of 22.7%, with no loss of tiger prawn. We also compared the numbers of seasnakes caught in 41 and 72 trawls during the spring trawling seasons of 2004 and 2005, respectively. The Yarrow Fisheye reduced the catches by a mean of 43.3%. Flume-tank tests of the Yarrow Fisheye showed that this device created a slow water-flow region extending over 2 m downstream from its position in the net, and close to where the catch accumulates. Finfish and seasnakes may be exploiting this slow water-flow region to escape via the eye, Although the reductions in fish and seasnake bycatch were excellent, we think they could be further improved by relating differences in fisheye position and localised water displacements, to design and rigging changes.

Subsampling multi-species trawl catches from tropical northern Australia: Does it matter which part of the catch is sampled?

Subsampling is a common method for estimating the abundance of species in trawl catches. However, the accuracy of subsampling in representing the total catch has not been assessed. To estimate one possible source of bias due to subsampling, we tested whether the position on trawler sorting trays from which subsamples were taken affected their ability to represent species in catches. This was done by sorting catches into 10 kg subsamples and comparing subsamples taken from different positions on the sorting tray. Comparisons were made after species were grouped into three categories of abundance, either 'rare', 'common' or 'abundant'. A generalised linear model analysis showed that taking subsamples from different positions on the sorting tray had no major effect on estimating the total numbers or weights of fish or invertebrates, or the total number of fish or invertebrate taxa, recorded in each position. Some individual taxa showed differences between positions on the sorting tray (11.5% of taxa ina three-position design; 25% in a five-position design). But consistent and meaningful patterns in the position of these taxa on the sorting tray could only be seen for the pony fish Leiognathus moretoniensis and the saucer scallop Amusium pleuronectes. Because most bycatch laxa are well mixed throughout the catch, subsamples can be taken from any position on trawler sorting trays without introducing bias.

Handling of Two Tropical Australian Sharks to Improve Quality and to Identify the Cause of Tough Texture

Sharks caught in tropical Australian waters occasionally exhibit tough texture. Two species of Carcharinid shark, originally known as the sorrah shark (Carcharinus sorrah) and the black spot shark (Carcharinus tilstoni), compose the majority of the catch. Experiments were conducted to identify the cause of tough texture and to improve the overall quality of the catch. The possibility that a cold shock reaction may occur was investigated by observing the contraction of fillets under a range of temperature conditions before freezing. The effect of on-board handling practices were evaluated using frozen shark fillets, which had been stored prior to freezing in refrigerated seawater at different rigor stages, temperatures and times as trunks. Fillets were analyzed for nucleotides, lactate, thaw pH, sarcomere length and raw and cooked shear force values. The existence of thaw rigor was also investigated. There was little difference in the texture between the individual strips of a fillet exposed to different temperatures but there were significant differences between individual sharks. A cold shock reaction could not be demonstrated in these species. The main influences on texture were of biological origin. The species, sex and size were found to have significant links with texture of fillets. The quality of the fillets deteriorated quicker during the warmer season and were at their worst if the trunks were kept on deck till post-rigor or held in 15 degree C refrigerated seawater before freezing

Volatile components associated with bacterial spoilage of tropical prawns

Analysis of headspace volatiles by gas chromatography/mass spectrometry from king (Penaeus plebejus), banana (P. merguiensis), tiger (P. esculentus/semisulcatus) and greasy (Metapenaeus bennettae) prawns stored in ice or ice slurry, which is effectively an environment of low oxygen tension, indicated the presence of amines at the early stages of storage (less than 8 days) irrespective of the nature of the storage media. Esters were more prevalent in prawns stored on ice (normal oxygen conditions) at the latter stages of storage (more than 8 days) and were only produced by Pseudomonas fragi, whereas sulphides and amines occurred whether the predominant spoilage organism was Ps.fragi or Shewanella putrefaciens. The free amino acid profiles of banana and king prawns were high in arginine (12–14%) and low in cysteine (0.1–0.17%) and methionine (0.1–0.2%). Filter sterilised raw banana prawn broth inoculated with a total of 15 cultures of Ps. fragi and S. putrefaciens and incubated for two weeks at 5°C, showed the presence of 17 major compounds in the headspace volatiles analysed using gas chromatography/mass spectrometry (GC/MS). These were mainly amines, sulphides, ketones and esters. Principal Component Analysis of the results for the comparative levels of the volatiles produced by pure cultures, inoculated into sterile prawn broth, indicated three subgroupings of the organisms; I, Ps. fragi from a particular geographic location; II, S. putrefaciens from another geographic location; and III, a mixture of Ps. fragi and S. putrefaciens from different geographic locations. The sensory impression created by the cultures was strongly related to the chemical profile as determined by GC/MS. Organisms, even within the same subgrouping classified as identical by the usual tests, produced a different range of volatiles in the same uniform substrate.