There is more to grass pollinosis than grass pollen and pollen counts. Part 3 of 3.
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BACKGROUND AND AIMS: It is well documented that C(4) grasses have a shorter distance between longitudinal veins in the leaves than C(3) grasses. In grass leaves, however, veins with different structures and functions are differentiated: large longitudinal veins, small longitudinal veins and transverse veins. Thus, the densities of the three types of vein in leaves of C(3) and C(4) grasses were investigated from a two-dimensional perspective. METHODS: Vein densities in cleared leaves of 15 C(3) and 26 C(4) grasses representing different taxonomic groups and photosynthetic subtypes were analysed. KEY RESULTS: The C(4) grasses had denser transverse veins and denser small longitudinal veins than the C(3) grasses (1.9 and 2.1 times in interveinal distance), but there was no significant difference in large longitudinal veins. The total length of the three vein types per unit area in the C(4) grasses was 2.1 times that in the C(3) grasses. The ratio of transverse vein length to total vein length was 14.3 % in C(3) grasses and 9.9 % in C(4) grasses. The C(3) grasses generally had greater species variation in the vascular distances than the C(4) grasses. The bambusoid and panicoid C(3) grasses tended to have a denser vascular system than the festucoid C(3) grasses. There were no significant differences in the interveinal distances of the three vein types between C(4) subtypes, although the NADP-malic enzyme grasses tended to have a shorter distance between small longitudinal veins than the NAD-malic enzyme and phosphoenolpyruvate carboxykinase grasses. CONCLUSIONS: It seems that C(4) grasses have structurally a superior photosynthate translocation and water distribution system by developing denser networks of small longitudinal and transverse veins, while keeping a constant density of large longitudinal veins. The bambusoid and panicoid C(3) grasses have a vascular system that is more similar to that in C(4) grasses than to that in the festucoid C(3) grasses.
BACKGROUND: In the subtropical climate of South Africa, grasses of the subfamily Panicoideae are predominant. Bermuda grass has previously been shown to be an important local allergen, and immunoglobulin E (IgE) epitopes of Bermuda grass extracts are known to be distinct from those of the Pooid pollen extracts. Following our demonstration of sensitivity in 43% of patients grass-allergic to the Panicoid, Kikuyu grass, we have studied the closely related buffalo grass, Stenotaphrum secundatum, indigenous to the Western Cape region, the east coast of Africa and the oceanic islands such as Mauritius; and Eragrostis, another common indigenous grass with a wide distribution. OBJECTIVE: To partially characterise the allergens of buffalo pollen, and examine its immunological relationships with local common grasses such as Eragrostis and Kikuyu. METHODS: Grass-allergic patients were evaluated clinically, and skin prick tests (SPTs) and radio-allergosorbent tests (RASTs) to Bermuda and grass mix were performed. Sera of timothy grass-sensitive patients from Belgium were also included in this study. Pollen extract from buffalo grass was characterised by specific IgE binding by means of immunoblotting and enzyme-linked immunosorbent assay (ELISA). Cross-reactivity between the grasses was studied by means of inhibition of IgE binding. RESULTS: More than 90% of grass-sensitive patients were found to have IgE antibodies to Buffalo and Eragrostis pollen. Inhibition of ELISA and immunoblots revealed that extracts of these grass pollens could significant inhibit IgE binding to the local grass pollens, Kikuyu, buffalo, Eragrostis and Bermuda on solid phase, but 100% inhibition was never achieved, indicating that cross-reactive but also unique epitopes are present. We also identified a subset of patients with negative RASTs to Bermuda, and minimal inhibition by Bermuda pollen extract. CONCLUSION: Buffalo and Eragrostis are important aeroallergens in the Cape, dispersed during the long dry, windy summer. Our data suggest that the local grasses are major sensitisers, and that South African diagnostic panels should include extracts of buffalo and Eragrostis grasses.
BACKGROUND: Immediate skin testing is generally the preferred method for establishing the presence of allergy in clinical practice. There is no agreement, however, as to whether intradermal testing should be routinely performed if skin prick test results are negative. PURPOSE: The study was done to address the value of intradermal skin testing in the diagnosis of clinically significant sensitivity to grass pollen in patients exhibiting negative skin prick test responses to timothy extract. METHODS: Four groups were studied. Group I had a history of seasonal allergic rhinitis, negative skin prick test responses to timothy and Bermuda grass, but positive intradermal skin test responses to timothy grass. Group II had a history of seasonal allergic rhinitis and positive skin prick test responses to timothy grass. Group III had a history of seasonal allergic rhinitis but had negative responses to both prick and intradermal testing with timothy and Bermuda grass. Group IV had no history of rhinitis, had negative responses to skin testing with a panel of locally important allergens, as well as Bermuda and timothy grass, and had a serum IgE value of less than 20 IU/ml. Clinical sensitivity to grass was assessed by two methods: (1) nasal challenge with threefold increasing amounts of timothy pollen performed out of the pollen season and (2) correlation of subjects' daily symptom and medication scores with daily grass pollen counts during the grass pollen season. RESULTS: On the basis of nasal challenge with timothy grass, pollen allergic reactions were present in 11% of group I, 68% of group II, 11% of group III, and 0% of group IV. As determined by correlation of symptoms during the grass pollen season with grass pollen counts, 22% of group I, 64% of group II, 21% of group III, and 0% of group IV were considered allergic. If both criteria were required for a diagnosis of clinical allergy to grass, the percent positive was 0 for group I, 46 for group II, 0 for group III, and 0 for group IV. CONCLUSION: Under the conditions of this study the presence of a positive intradermal skin test response to timothy grass (1000 AU/ml) in the presence of a negative skin prick test response to timothy grass (100,000 AU/ml) did not indicate the presence of clinically significant sensitivity to timothy grass, and by inference, to other cross-reacting grasses.
RAST inhibition was employed to determine the allergenic relationship of five common western grasses (brome, quack, western wheat, salt, and grama), three southern grasses (Bermuda, Johnson, and Bahia), and six common northern grasses (timothy, June, rye, red top, meadow fescue, and sweet vernal). The patterns of cross-allergenicity suggested by these studies were compared to those suggested by the botanical classification of these grasses. There was significant cross-allergenicity among the grasses studied. Brome, western wheat, and quack grasses demonstrated RAST inhibition patterns similar to the northern grasses. Salt and grama grasses exhibited close allergenic relationships to Bermuda grass, which was allergenically distinct from the northern grasses. Johnson grass appeared to share allergenicity with both the northern grasses and Bermuda grass. Bahia shared allergenicity with the northern grasses, but also possessed some unique allergens. Timothy and Bermuda grass appeared to account for all of the RAST-inhibiting activity contained in the other grasses studied with the possible exception of Bahia grass.
The increasing CO2 concentration in Earth's atmosphere is expected to cause a greater decline in the nutritional quality of C3 than C4 plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C3 plants. These hypotheses were tested by growing the grasses Lolium multiflorum C3) and Bouteloua curtipendula C4) at ambient (370 ppm) and elevated (740 ppm) CO2 levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant x CO2 treatment combinations. As expected, the nutritional quality of the C3 grass declined to a greater extent than did that of the C4 grass at elevated CO2; protein levels declined in the C3 grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C3 grass grown under elevated CO2. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C3 grass under elevated CO2. Consumption rates of the C3 and C4 grasses were also similar, demonstrating a lack of compensatory feeding on the C4 grass. We also examined the relative efficiencies of nutrient utilization from a C3 and C4 grass by M. sanguinipes to test the basis for the C4 plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C4 grass than from the C3 grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C3 grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C3 grass and the better growth performance of M. sanguinipes on this grass at both CO2 levels, we conclude that C3 grasses are likely to remain better host plants than C4 grasses in future CO2 conditions.
BACKGROUND: Pollinosis, caused by grasses of the Poaceae family, is a problem worldwide. The relative importance of grass groups 1 and 5 major allergens is well established. However, not much is known about the recognition of these allergens by T cells and whether this T-cell reactivity correlates with skin reactivity and serum IgE levels. OBJECTIVES: The aim of this study was to characterize the cross-reactive, grass allergen-specific T-cell responses from patients allergic to grass and nonatopic individuals and to investigate whether these responses correlate with grass-specific IgE and skin reactivity. METHODS: Skin prick test wheal areas and grass-specific serum IgE levels were determined in all patients (n = 21) and nonallergic control donors (n = 20). Peripheral blood mononuclear cells were isolated and stimulated with grass allergen extracts (Phleum pratense, Poa pratensis, Lolium perenne) and immunoaffinity-purified group 5 allergens, and the production of type 1 and type 2 cytokines was determined in the patient group. RESULTS: Donors allergic to grass showed increased T-cell-proliferative responses to grass allergens compared with nonatopic control subjects. We find it interesting that the magnitude of the patients' T-cell responses could not be correlated with the individual skin prick test areas and specific serum IgE levels, and several patients with allergies to grass had group 5-specific T-cell responses in the absence of group 5-specific IgE. The absence of a correlation between T-cell proliferation and IgE levels or skin prick test results may in part be explained by the finding that patients predominantly produced IL-5 in response to Phl p 5, the major allergen, and predominantly IFN-gamma in response to Phl p extract. In general, all donors responded equally well to all three grasses. Additional experiments with Phl p 5-specific T-cell lines indicated that the equal proliferation of peripheral blood mononuclear cells to all three species is the direct result of cross-reactivity. CONCLUSIONS: Grass allergen-specific T-cell responses are highly cross-reactive, and patients with allergies exhibit higher responses than nonallergic donors, suggesting that T cells are involved in the allergic reaction to grass group 5 allergens. However, group 5-specific T-cell responses are also found in donors without group 5-specific IgE, and the patients' grass-specific T-cell responses and cytokine production do not correlate to skin reactivity or to concentrations of grass-specific IgE.
BACKGROUND: The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen source in the external environment of southern Australia. This study was performed to characterize the major recombinant allergens of canary grass pollen. It is anticipated that recombinant allergens may be useful in diagnosis and immunotherapy of grass pollen induced allergies. OBJECTIVE: To clone major canary grass pollen allergens and assess their nucleotide and amino acid sequence homologies with other grass pollen allergens. This sequence information may then be useful in T and B cell epitope mapping studies. METHODS: A canary grass pollen lambda gt11 cDNA expression library was constructed and screened with sera of grass-pollen-sensitive patients. IgE-reactive clones were isolated, sub-cloned into Escherichia coli, sequenced and, along with the deduced amino acid sequences, compared with other sequences in nucleotide and amino acid databases. RESULTS: One of the clones encoded the group 1 allergen of canary grass pollen, Pha a 1, with a deduced amino acid sequence identity of 88.8% with Lol p 1, from rye-grass pollen, 88.1% with Hol l 1, from velvet grass pollen and 86.6% with Phl p 1, from timothy grass pollen. The other clones (e.g. clones, 5, 14, 28, 29) encoded polymorphic forms of Pha a 5. These polymorphic forms showed between 60.6-95.5% nucleotide and 40.1-81.7% deduced amino acid sequence identities with each other. Moreover, they shared significant sequence identity with other group 5 allergens from rye-grass, timothy and Kentucky bluegrass pollens. CONCLUSIONS: Group 1 and four isoforms of group 5 allergens of canary grass pollen have been cloned and upon sequencing demonstrated strong nucleotide and amino acid sequence identities with other group 1 and 5 grass pollen allergens.
The invasion of African grasses into Neotropical savannas has altered savanna composition, structure and function. The projected increase in atmospheric CO(2) concentration has the potential to further alter the competitive relationship between native and invader grasses. The objective of this study was to quantify the responses of two populations of a widespread native C(4) grass (Trachypogon plumosus) and two African C(4) grass invaders (Hyparrhenia rufa and Melinis minutiflora) to high CO(2) concentration interacting with two primary savanna stressors: drought and herbivory. Elevated CO(2) increased the competitive potential of invader grasses in several ways. Germination and seedling size was promoted in introduced grasses. Under high CO(2), the relative growth rate of young introduced grasses was twice that of native grass (0.58 g g(-1) week(-1) vs 0.25 g g(-1) week(-1)). This initial growth advantage was maintained throughout the course of the study. Well-watered and unstressed African grasses also responded more to high CO(2) than did the native grass (biomass increases of 21-47% compared with decreases of 13-51%). Observed higher water and nitrogen use efficiency of invader grasses may aid their establishment and competitive strength in unfertile sites, specially if the climate becomes drier. In addition, high CO(2) promoted lower leaf N content more in the invader grasses. The more intensive land use, predicted to occur in this region, may interact with high CO(2) to favor the African grasses, as they generally recovered faster after simulated herbivory. The superiority of invader grasses under high CO(2) suggests further increases in their competitive strength and a potential increased rate of displacement of the native savannas in the future by grasslands dominated by introduced African species.
BACKGROUND: Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization. OBJECTIVE: The aim of the study was to investigate whether mixtures of grass 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 = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5. RESULTS: Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results. CONCLUSION: One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected. Around 80% of the IgE response to grass pollen is directed to these major allergens. Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts.
Six grass pollen allergens have been individually polymerized. We had previously polymerized mixed grass pollen allergens. However, since every patient does not react to every grass, we sought to polymerize individual grasses that could subsequently be mixed into a preparation based on a patient's skin reactivity. As demonstrated by Sepharose 4-B chromatography of individual grass monomers and polymers, all six grasses were polymerized. Polymerized grass preparations as compared with monomer preparations demonstrated a 10(3)- to 10(5)-fold reduction in allergenicity as determined by cutaneous end point titer. That grass polymer contains the great majority of clinically important allergenic determinants was demonstrated by the ability of polymer to inhibit 78% IgE binding against crude grass in a pool of untreated grass-sensitive patients. Its antigenic similarity to crude grasses is further shown by the ability of polymer to inhibit 85% of IgG binding against crude grass in a pool of patients treated with usual crude grass extracts.
BACKGROUND: Polygalacturonases were recently identified as important grass pollen allergens and designated group 13 allergens. The objective of the present study was to investigate the presence of group 13 grass pollen allergens in different grass species, their release and ultrastructural location in dry and hydrated grass pollen. METHODS: Nitrocellulose-blotted allergen extracts from 12 wild and cultivated grass genera were probed with a rabbit antiserum raised against purified recombinant timothy grass pollen allergen, Phl p 13. The release kinetics of Phl p 13 from timothy grass pollen hydrated for 0.5 min to 3 h were analyzed by immunoblotting. Phl p 13 was localized in dry and hydrated grass pollen grains by immunogold field emission scanning and transmission electron microscopy. RESULTS: Group 13 allergens were detected in all 12 wild and cultivated grass genera representing the major subfamilies of the Poaceae. Ultrastructurally, the allergen was located in the wall and in the cytoplasm of timothy grass pollen grains. In the cytoplasm, Phl p 13 was associated with polysaccharide particles and as yet undescribed stacks of microtubule-like structures. After hydration in rain water, pollen grains expel cytoplasmic particles of respirable size containing Phl p 13, which becomes detectable in aqueous supernatants already after 0.5 min. CONCLUSIONS: Group 13 allergens represent one set of marker allergens which specifically occur in pollen of the major grass subfamilies and are rapidly released in association with respirable particles after pollen hydration. They may be considered as environmental markers for grass pollen exposure and group 13-specific IgE antibodies as immunological markers for genuine grass pollen sensitization.
In this study, a heterologous radioimmunoassay (RIA) for grass carp GH has been validated and used to monitor the kinetics of GH release from perifused grass carp pituitary cells. To establish the anatomical specificity of GH antiserum used in this RIA, immunohistochemical staining was performed in grass carp pituitary sections. Somatotrophs recognized by this GH antiserum were located mainly in the proximal pars distalis without overlapping with gonadotrophs located in the same area or with lactotrophs located in the rostral pars distalis. The immunoreactivity of somatotrophs was abolished by preabsorbing GH antiserum with purified grass carp GH, suggesting that the possibility of a cross-reactivity of antiserum with other grass carp pituitary hormones is unlikely. Using 125I-labeled carp GH as the RIA tracer, parallelism was observed among the displacement curves of grass carp GH standard, grass carp serum, and culture medium conditioned by grass carp pituitary cells, suggesting that this RIA can be used to quantitate grass carp GH levels in biological samples. Using an in vitro column perifusion system, a superactive gonadotropin-releasing hormone (GnRH) analog (d-Arg6, Pro9-NEt)-sGnRH(sGnRHa, 0.3-30 nM), dopamine (DA, 0.1-10 muM), and the nonselective DA agonist apomorphine (0.1-10 muM) stimulated GH release from grass carp pituitary cells in a dose-dependent manner. The GH-releasing effect of DA was mimicked by the D1 agonists SKF38393 (0.1-10 muM) and SKF77434 (0.1-10 muM), but not by the D2 agonist LY171555 (3 muM). In addition, the GH response to DA (1 muM) was blocked by the D1 antagonist SCH23390 (5 muM) but not by the D2 antagonist (+/-) sulpiride (5 muM), suggesting that the GH-releasing action of DA is mediated through receptors resembling mammalian D1 receptors. Somatostatin-14 (SRIF14, 0.01-100 nM), unlike sGnRHa and DA, induced a dose-dependent suppression on basal GH release. At a high dose (100 nM), SRIF14 also abolished the GH responses to sGnRHa (100 nM), DA (10 muM), and the D1 agonist SKF38393 (3 muM). These results, as a whole, provide evidence that GH release in the grass carp is under the direct regulation of GnRH, DA, and SRIF at the pituitary cell level. The present study also suggests that DA D1 receptors are present in grass carp pituitary cells mediating the GH-releasing action of DA.
BACKGROUND AND AIMS: Photorespiration occurs in C4 plants, although rates are small compared with C3 plants. The amount of glycine decarboxylase in the bundle sheath (BS) varies among C4 grasses and is positively correlated with the granal index (ratio of the length of appressed thylakoid membranes to the total length of all thylakoid membranes) of the BS chloroplasts: C4 grasses with high granal index contained more glycine decarboxylase per unit leaf area than those with low granal index, probably reflecting the differences in O2 production from photosystem II and the potential photorespiratory capacity. Thus, it is hypothesized that the activities of peroxisomal enzymes involved in photorespiration are also correlated with the granal development. METHODS: The granal development in BS chloroplasts was investigated and activities of the photorespiratory enzymes assayed in 28 C4 grasses and seven C3 grasses. KEY RESULTS: The NADP-malic enzyme grasses were divided into two groups: one with low granal index and the other with relatively high granal index in the BS chloroplasts. Both the NAD-malic enzyme and phosphoenolpyruvate carboxykinase grasses had high granal index in the BS chloroplasts. No statistically significant differences were found in activity of hydroxypyruvate reductase between the C3 and C4 grasses, or between the C4 subtypes. The activity of glycolate oxidase and catalase were smaller in the C4 grasses than in the C3 grasses. Among the C4 subtypes, glycolate oxidase activities were significantly smaller in the NADP-malic enzyme grasses with low granal index in the BS chloroplasts, compared with in the C4 grasses with substantial grana in the BS chloroplasts. CONCLUSIONS: There is interspecies variation in glycolate oxidase activity associated with the granal development in the BS chloroplasts and the O2 production from photosystem II, which suggests different potential photorespiration capacities among C4 grasses.